BSC 300 Exam 3

laminin is a heterotrimeric multi-adhesive matrix protein found in all basal laminae

-16 vertebrae laminin isoforms -coiled-coil region - 3 peptides covalently linked by several disulfide bonds -globular domains - bind to/crosslink adhesion receptors and various matrix components -five alpha subunit C-terminal globular LG domains - mediate calcium-dependent binding to cell-surface laminin receptors, including certain integrins, sulfated glycolipids, syndecan, and dystroglycan -inset: laminins assemble into a lattice via interactions between their N-terminal globular domains

insertion of tail-anchored proteins

-C-terminal tail-anchored proteins -no N-terminal SS -hydrophobic C-terminus - not available for membrane insertion until protein synthesis is complete and the protein has been released from the ribosome -step 1 - Sgt2/Get4/Get5 complex sequesters the nascent protein hydrophobic C-terminal tail anchor sequence and transfers it to Get3-ATP -step 2 - Get3-ATP-nascent protein complex docks onto the ER membrane dimeric Get1/Get2 receptor -step 3 - Get3 ATP hydrolysis and ADP release - releases nascent protein hydrophobic C-terminal tail into the Get1/Get2 receptor; Get1/Get2 receptor releases tail-anchor sequence into the ER membrane -step 4 - Get3 releases of ADP and binding ATP releases it from Get1/Get2

model for the generations of cell-cell adhesions

-CAM cis interactions - intracellular lateral interactions - forms lateral clusters within the plasma membrane, regions that form cis interactions vary among different CAMs -trans interactions - intercellular adhesive interactions - generate strong, velcro-like adhesions between the cells -trans and cis interactions are mutually reinforcing

translation on bound ribosomes

-ER signal sequence on N-terminus is bound by a cytoplasmic protein called the signal recognition particle (SRP) -SRP delivers the ribosomes/mRNA/peptide complex to the SRP receptor on the membrane of the RER - SRP is released -SRP receptor passes ribosomes to a protein translocator (translocon) that remains bound to the signal while peptide translation continues into the ER lumen -if this is a soluble protein once the C-terminus has passed through the translocator, the signal sequence is cleaved and the protein is released

targeting sequences on the cytosolic regions of cargo proteins bind to specific coat proteins

-KDEL: ER-resident soluble proteins, KDEL receptor in cis-golgi membrane, COPI -M6P: soluble lysosomal enzymes after processing in cis-golgi, M6P receptor in trans-golgi membrane, clathrin/AP1 -KKXX: ER-resident membrane proteins, COPI alpha and beta subunits, COPI

action of protein disulfide isomerase (PDI)

-PDI forms and regenerates protein cysteine disulfide bonds; active site - contains two closely spaced cysteines that are easily interconverted between the reduced dithiol form and the oxidized disulfide form -formation of disulfide bonds (electron transfers): substrate protein ionized cysteine thiol, substrate protein second ionized thiol, regeneration -rearrangement of improper disfulfide bonds - commonly misform between cysteine that occur sequentially in the amino acid sequence, while a polypeptide is still growing -reactions - repeated until the protein is folded into the thermodynamically most stable conformation

the transferring cycle operates in all growing mammalian cells

-RME endocytic pathway delivers iron to cells without dissociation of the transferrin-transferrin receptor complex in endosomes -transferrin protein: apotransferrin (no bound Fe3+, doesn't bind well at pH 7), ferrotransferrin (carries Fe3+ in blood), binds to transferrin receptor -step 1 - ferrotransferrin dimer with two Fe3+ binds to transferrin receptor at cell surface = pH 7 -step 2 - transferrin receptor cytoplasmic tail interaction with an AP2 adapter complex -step 3 - vesicle fuses with late endosome - acidic pH, become apotransferrin -step 4 - recycling receptor -step 5 - extracellular neutral pH destabilizes complex - released apotransferring

model for docking and fusion of transport vesicles with their target membranes

-Rab GTPases control docking of vesicles on target membranes -fusion of secretory vesicles with the plasma membrane - similar mechanism mediates all vesicle-fusion events: -step 1 - transport vesicle docking on appropriate target membrane - Rab protein tethered via a lipid anchor to a secretory vesicle binds to an effector protein complex on the target (plasma) membrane -step 2 - v-SNARE protein (VAMP - on vesicle) forms stable coiled-coil interactions with cognate t-SNAREs (on target membrane) cystolic domains; SNARE complexes - hold vesicle close to the target membrane -step 3 - fusion of the two membranes - drives dissociation of the SNARE complexes, freeing SNARE proteins for another round of vesicle fusion; Rab-GTP hydrolyzed to Rab-GDP - dissociates Rab from the Rab effector -step 4 - dissociation of the SNARE complexes requires the assistance of other proteins; NSF (regenerates snares), a hexamer of identical subunits, associates with a SNARE complex with the aid of alpha-SNAP; bound NSF then hydrolyzes ATP, releasing sufficient energy to dissociate the SNARE complex

Sec61alpha is a translocon component

-Sec61alpha translocon component; contacts nascent secretory proteins as they pass through the translocon into the ER lumen -neither SRP nor Sec61 hydrolyzes ATP - translocation through the translocon is driven by translation elongation

biosynthesis of the oligosaccharide precursor

-a preformed N-linked oligoaccharide is added to many proteins in the rough ER -oligosaccharide precursor containing 14 residues - preformed on dolichol phosphate embedded in ER membrane -dolichol phosphate - hydrophobic polyisoprenoid lipid containing 75-95 carbon atoms -steps 1-3 - two n-acetylglucosamine (GlcNAc) and 5 mannose residues - high-energy pyrophosphate linkages added one at a time on the ER membrane cytosolic face -step 4 - the seven-residue dolichol pyrophosphoryl intermediate is flipped to the ER membrane luminal face -steps 5-6 - additional sugars by ER enzymes

the endocytic pathway

-after internalization, vesicle-bound materials are transported in vesicles and tubules known as endosomes -early endosomes are located near the periphery of the cell, it sorts materials and sends bound ligands to the late endosomes, acidic pH maintained by H+ATPase, separation of ligand from receptor -materials sorted: house-keeping receptors recycled to cell membrane, ligands and dissolved solutes transported to late endosomes - endosomal carrier vesicles formed from early endosomes -late endosomes are near the nucleus, also known as multivesicular bodies (MVBs), destination of lisosomal enzymes included in golgi vesicles, some receptors are recylced to TGN (M6P) -lysosomal enzyjmes and endocytic material delivered to lysosomes - several routes: maturation of late endosomes into lysosomes, fusion of late endosomes with lysosomes, and transport from late endosomes to lysosomes in vesicles

overview of major protein-sorting pathways in eukaryotes

-all nuclear DNA-encoded mRNAs - translated on cytosolic ribosomes -protein targeting/sorting: delivery of newly synthesized proteins to their proper cellular destinations, occurs specifically during translation or soon after synthesis

protein import into the mitochondrial matrix

-amphipathic N-terminal targeting sequences target proteins to the mitochondrial matrix: N-terminal 20-50 amino acids - amphipathic alpha-helical conformation (using the backbone), positively charged and hydrophobic amino acids predominate on opposite sides of the helix -signal sequence recognized by a receptor protein in the transport outer membrane (TOM) complex -signal sequence inserted through TOM complex interacts with receptor in the transporter inner membrane (TIM) complex -protein translocation driven by the hydrogen ion gradient -matrix chaperone proteins bind polypeptide chain; helps it refold -transport requires energy (different from secretory pathway), hydrolysis of ATP and gradient provide energy -mechanism: precursor proteins synthesized on cytosolic ribosomes -step 1 - maintained in an unfolded state by bound chaperones, such as cytosolic Hsp70 - hydrolyze ATP to ADP and clamp on protein to prevent folding -step 2 - matrix-targeting sequence (MTS) binds to outer membrane imort receptor near a rare "contact site" with the inner membrane -step 3 - MTS inserted into outer membrane translocon (Tom - translocon of the outer membrane) -protein can be in TIM and TOM complexes at the same time when they are close enough -step 4 - translocating protein moves through tom and inserts into inner membrane translocon (tim - translocon of the inner membrane) -step 5 - protein translocates through tim -binding by matrix Hsp70 helps drive import into the matrix -MTS removed by a matrix protease -step 6 - Hsp70 ATP hydrolysis released newly imported protein -step 7 - protein folds into its mature, actie conformation within the matrix -folding of some proteins depends on other matrix chaperones -don't need signal once it's in the matrix -3 energy inputs required for import: 1. cytosolic Hsp70 - expends ATP energy to maintain precursor proteins in an unfolded state for translocation through translocons 2. matrix Hsp70 anchored to the Tim44 protein - may act as a molecular motor to pull the protein into the matrix 3. H+ electrochemical gradient (proton-motive force) across the inner membrane - may electrophorese proteins into the matrix

GPI-anchored proteins

-amphipathic glycosylphosphatidylinositol (GPI) molecule - anchor covalently attached protein in membrane (protein outside cell in plasma membrane) -GPI molecule from yeast - hydrophobic portion (fatty acyl chains), hydrophilic portion (carbohydrate residues and phosphate groups), length of acyl chains and carbohydrate moieties may vary in other species -GPI-anchored protein formation: protein - synthesized and inserted into the ER membrane like a type I transmembrane protein; specific transamidase - cleaves the precursor protein within the exoplasmic-facing domain, near the stop-transfer anchor sequence, covalently links new C-terminus to the terminal amino group of a preformed GPI anchor

hemidesmosomes

-basal attachments of epithelial cells to the basement membrane in vivo -contain a dense plaque with filaments consisting of keratin -keratin filaments are linked to the ECM by membrane-spanning integrins -integrins bound to basal lamina laminin -blisters are the result of regionalized loss of hemidesmosomes

a basal lamina separates epithelial cells and some other cells from connective tissue

-basal lamina - foundation for assembly of cells into most tissues, a meshwork of filamentous proteins that associates with the plasma membrane and connective tissue collagen fibers -cells and underlying connective tissue (EM) -skeletal muscle plasma membrane, basal lamina, and surrounding connective tissue collagen fibers -functions in organizing cells into tissues and distinct compartments, protecting cells, repairing tissues, forming permeability barriers, and guiding migrating cells during development -one side - linked to cells by adhesion receptors including hemidesmosome integrins that bind to laminin in the basal laminal -other side - anchored to adjacent connective tissue by a layer of collagen fibers embedded in a proteoglycan-rich matrix

synthesis and secretion of collagen

-because of its size, ECM collagen cannot assemble as fibrils/fibers intracellularly; instead, when synthesized it contains N- and C-termini that inhibit these associations -this immature collagen is pre-procollagen -extracellular procollagen proteinases cleave these termini allowing mature collagen polypeptides to self-assemble -to live and work within such a matrix of proteins, cells are responsible for tissue growth, repair, and renewal, and must be able to migrate and move within the ECM -important for fibroblasts and immune cells like macrophages -such cells secrete enzymes called matrix proteinases that degrade ECM proteins including collagen -inappropriate and overexpression of such proteinases is a hallmark of many diseases like arthritis and cancer -fibroblasts and osteoblasts do not simply produce collagen but arrange and modify it, influencing tissue structure an promoting wound healing -connective tissue is often an orderly array of collagen fiber layers often oriented at right angles to provide greatest structural support -fibroblasts actively migrate on the collagen they produce rearranging and promoting organized assembly

proteins unfold to enter mitochondria/chloroplasts

-both organelles contain their own genome, produce their own ribosomes, and synthesize some of their own proteins -however, the majority of proteins found within these organelles are derived from nuclear genes and therefore must be imported into the organelle after being synthesized in the cytoplasm -N-terminal organelle-specific signal sequence

interactions of cells with other cells

-cadherins - glycoproteins that mediate Ca2+-dependent cell-cell adhesion in adherens junctions, many types: epithelial, neural, and placental

model for formation of secretory vesicles

-cargo not tagged for separation secreted by default -included in vesicles when TGN fragments -plasma membrane proteins included in vesicle membrane -densely packed secretory proteins concentrated by aggregation and trapped in vesicles

major families of cell-adhesion molecules (CAMs) and adhesion receptors

-cell-adhesion molecules bind to one another and to intracellular proteins -CAM cytoplasmic domains - often associate with adapter proteins that link them to the cytoskeleton/signaling pathways -form homophilic or heterophilic interactions 1. cadherins: forms Ca2+-dependent extracellular homophilic interactions with E-cadherins on adjacent cells 2. IgCAMs: members of the immunoglobulin (Ig) superfamily, form homophilic or heterophilic linkages 3. integrins: heterodimeric (alpha and beta chains), adhesion receptors - bind to very large, multi-adhesive matrix proteins such as fibronectin 4. selectins: contain a carbohydrate-binding lectin domain that recognizes specialized sugar structures on adjacent cell glycoproteins/glycolipids, often form higher-order oligomers within the plane of the plasma membrane

overview of major cell-cell and cell-matrix adhesive interactions

-cell-cell and cell-matrix adhesions aggregate cells into distinct tissues to cooperatively perform common functions -intestinal epithelial tissue cells: 1. apical (upper) surface - packed with fingerlike microvilli that project into the intestinal lumen 2. basal (lower) surface - adhered to basal lamina 3. extracellular matrix (ECM) associated with epithelial cells - usually organized into various interconnected layers such as basal lamina and ECM (connecting fibers and connective tissue in which large, interdigitating ECM macromolecules bind to one another 4. cell-adhesion molecules (CAM) - bind to CAMs on other cells for cell-cell adhesion 5. cell-matrix adhesion - adhesion receptors bind to ECM components -cell-surface adhesion molecules - most are integral membrane proteins - cytosolic domains bind to multiple intracellular adapter proteins -adapter proteins - directly or indirectly link CAMs to the cytoskeleton (actin or intermediate filaments) and intracellular signaling pathways -several signal transduction pathways are activated through cell-cell and cell-ECM interactions: outside-in (from CAMs and bound extracellular macromolecues to the cytoplasm) and inside-out (from the cytoplasms through adapter proteins to CAMs and bound extracellular macromolecules) -junctions - localized aggregates of CAMs/adhesion receptors from various types of cell junctions holding tissues together and facilitating communication between cells and their environment -tight junctions - specific to the microvilli, prevents/regulates diffusion of many substances through the extracellular spaces between the cells, prevents protein/lipid exchange between the apical and basolateral regions of the plasma membrane -gap junctions - cluster of connexon channels, allow movement of small molecules and ions between cytosols of adjacent cells -adherens junctions: belt desmosome (subjacent to the tight junction, adheres cell to all neighboring cells), desmosomes (link intermediate filament cytoskeletons between neighboring cells), hemidesmosomes (link intermediate filament cytoskeleton to basal lamina ECM)

the initial staged of receptor-mediated endocytosis of low-density lipoprotein (LDL) particles

-cells take up lipids from the blood in the form of large, well-defined lipoprotein complexes -all classes of lipoproteins have the same general structure: shell composed of apolipoprotein and a phospholipid monolayer containing cholesterol, and a hydrophobic core composed mostly of cholesteryl esters/triglycerides -LDL particle - contains only a single molecule of one type of apolipoprotein (ApoB) wrapped around the outside of the particle -step 1 - cell-surface LDL receptor: extracellular domain binds LDL particle to ApoB protein tightly at extracellular neutral pH, cytosolic tail NPXY sorting signal - interaction with AP2 complex -step 2 - clathrin-coated pit containing receptor LDL complexes pinches it off -step 3 - vesicle fuses with endosome -step 4 - LDL receptor recycled to the cell surface -step 5 - late endosome fusion with lysosome -LDL receptor at neutral pH (as at the cell surface): ligand binding arm seven cysteine-rich repeats - tightly bind LDL ApoB-100 -LDL receptor at acidic pH (as in endosome): beta-propeller domain histidine residues - become protonated, positively charged propeller domain binds negatively charged ligand-binding domain residues - cause release of the LDL particle

transporting proteins to chloroplast thylakoids

-chloroplasts have 6 compartments: outer and inner membranes, thylakoid membrane surrounding thylakoid lumens -protein directed to compartments by targeting signals -two of four pathways for transporting proteins from the cytosol to the thylakoid lumen: all proteins contain and N-terminal stromal-import sequence; unfolded precursor - translocated through outer (toc) and inner membrane (tic) translocons; no proton-motive force across either the outer or inner membrane - translocation required ATP energy -left SRP-dependent pathway: 1. translocation through toc and tic: cleavage of the N-terminal stromal-import sequence by a stromal protease - uncovers thylakoid-targeting sequence at new N-terminus; chaperones maintian proteins unfolded in the stromal space 2. thylakoid targeting- sequence bound by proteins closely related to the bacterial SRP, SRP receptor, and SecY translocon 3. thylakoid-targeting sequence - removed by thylakoid lumen endoprotease - protein folds into mature conformation -right pH-dependent (pmf-dependent) pathway - proteins that bind metal cofactors: 1. translocation through toc and tic 2. metal-binding proteins fold and bind complex redox cofactors in the stroma 3. thylakoid-targeting sequence - two N-terminal closely-spaced arginines: twin arginine signal induces twin-arginine translocation (tat) proteins to oligomerize into pore-like structures 4. transport f folded protein through tat pore

role of the KDEL receptor in retrieval of ER-resident luminal proteins from the golgi

-cis-golgi network - ER-to-golgi intermediate sorting compartment -soluble ER luminal resident proteins: 1. function in the ER to modify newly synthesized proteins 2. several types contain a C-terminal KDEL ER-targeting sequence 3. may be missorted to the cis-golgi network - nonspecifically incorporated in a COPII vesicle fluid phase, fusion of the vesicle delivers the ER resident protein to the cis-golgi network -KDEL receptor can't interact in basic conditions -retrieval system prevents depletion of ER luminal proteins needed for proper folding of newly made secretory proteins -addition of KDEL sequence to C-terminus of protein normally secreted becomes localized in ER

clathrin-coated pits and vesicles

-clathrin plays no part in selecting the specific cargo for the vesicle, their job is solely to pinch the vesicle from the membrane -the geometric association of clathrin proteins leads to spherical formation of the vesicle -once formed, clathrin is released form the vesicle, exposing receptor proteins that direct where the vesicle will be delivered -ARF GTPases assemble clathrin coat -triskelion structure -the GTPase dynamin plays an essential role in releasing the clathrin-coated vesicle from the membrane -it wraps around the membrane stalk and GTP hydrolysis causes conformational change that severs the membrane

gels of polysaccharides resist compression

-collagen provides tensile strength, other molecules provide cushioning and resistance to compression -glycosaminoglycans (GAGs): negatively charged polysaccharide chains of repeated disaccharide units (units of disaccharides may vary) -GAGs are covalently linked to core proteins to form proteoglycans -typically, many GAG chains are attached to a core protein, which may then be linked end to end to form a huge molecule -GAGs can assemble as complex chains -the negatively charge molecules attract cations like Na+ which draws water into the matrix forming a gel cushioning to the embedded cells -ratio of collagen to GAGs depends on the connective tissue type -dense, compact tissue like bones and tendons - matrix is mostly collagen; soft tissue like the interior of the eyes is mostly GAG matrix -proteoglycans additionally can regulate diffusion of signaling molecules, and in some cases are necessary cofactors in signal molecule binding to target receptors -proteoglycans also contribute to guiding cell migration by providing chemical cues

ribosomes and protein translation

-common pool of ribosomes synthesizes both cytosolic and ER destined proteins -ER signal sequence directs free ribosomes to dock on the ER membrane -both free and bound ribosomes translate mRNA in polyribosome complexes: a single mRNA bound by multiple ribosomes at various stages of translation

rough ER

-composed of a network of flattened sacs (cisternae) -continuous with the outer membrane of the nuclear envelope and also has ribosomes on its cytosolic surface -different types of cells have different ratios of the two types of ER depending on activities of the cell -attached ribosomes actively synthesize proteins

the endoplasmic reticulum (ER)

-comprises a network of membranes that penetrate much of the cytoplasm -like other organelles, the ER is highly dynamic undergoing continual turnover and reorganization -divided into two subcompartments: rough ER and smooth ER -in addition, the composition of the lumenal or cisternal space inside ER membranes is different from the surrounding cytosolic space -entry point for proteins destined for golgi, endosomes, lysosomes, plasma membrane, and for export - such proteins do not re-enter cytosol -protein synthesis begins on cytoplasmic free ribosomes that then associate with ER membrane after reading ER signal sequence, i.e. bound ribosomes -includes both soluble and membrane bound proteins

desmosomes

-contain two specialized cadherins - desmoglein and desmocollin -cytosolic domains - distinct from those in classical cadherins, bind adapter proteins - including plakoglobin, desmoplakins, and plakophilins - attach to sides of intermediate filaments -connect two cultured differentiated human keratinocytes (EM): bundles of intermediate filaments radiate from the two darkly staining cytoplasmic plaques that line the inner surface of the adjacent plasma membranes

structure of the signal recognition particle (SRP)

-cotranslational translocation - initiated by SRP and SRP receptor GTP-hydrolyzing proteins -cytosolic ribonucleoprotein particle - six proteins bound to a 300-nucleotide RNA scaffold -P54 subunit - M domain methionine and other hydrophobic amino acid in cleft residues binds to the signal sequence hydrophobic core -other polypeptides interact with the large (60S) ribosomal subunit, forming an mRNA-ribosome-partially synthesized nascent protein-SRP complex -GTP and receptor-binding domain - binds complex to an SRP receptor in the ER membrane -signal-sequence-binding domain: (bacterial Ffh protein - homologous to the P54 portion that binds ER signal sequences in eukaryotes); large cleft lines with hydrophobic amino acids - binds to signal sequence hydrophobic core -GTP and receptor-binding domain: structure of GTP bound to FtsY (the archaeal gomolog of the alpha subunit of the SRP receptor) and Ffh subunits

model for the role of Sar1 in the assembly and disassembly of COPII coat

-cycle of GEF-activated GTP binding and GAP-activated GTP-hydrolysis controls assembly and disassembly of vesicle coats -mechanisms are well conserved between the Sar1 G-protein (COPII) and ARf G-protein (COPI and Clathrin) -step 1 - Sar1 membrane binding, GTP exchange -step 2 - COPII coat assembly -step 3 - GTP hydrolysis -step 4 - coat disassembly

modification of N-linked oligosaccharides are used to monitor folding and for quality control

-dislocation: misfolded secretory proteins - recognized by specific ER membrane proteins and targeted for transport from the ER lumen into the cytosol for degredation -proteins that cannot fold properly - retained in the ER for longer times, eventually undergo mannose trimming

vesicle-mediated protein trafficking from the trans-golgi network

-distal sorting compartment -sorts proteins into five different types of vesicles for transport to the plasma membrane, endosomes, and lysosomes: 1. COPI vesicles - retrograde transport of golgi enzymes to the trans-golgi (cisternal progression process) 2. AP complex vesicles - may have clathrin coat, transport lysosomal enzymes directly to lysosomes 3. clathrin-coated (+AP2) vesicles - transport lysosomal enzymes to late endosomes for eventual delivery to lysosomes 4. constitutive secretory vesicles (unknown coat) - transport constitutively secreted proteins and plasma membrane proteins to the plasma membrane, cargo proteins include ECM proteins, blood proteins, and immunoglobulins 5. regulated secretory vesicles (unknown coat) - store and process secreted proteins until signaled to fuse with the plasma membrane to secrete the proteins, cargo proteins include digestive enzymes and peptide hormones

the dystrophin glycoprotein complex (DGC) in skeletal muscle cells

-dystrophin glycoprotein complex (DGC) - 3 subcomplexes -dystrophin - adapter protein - links the actin cytoskeleton to alpha-dystrobrevin and the sarcoglycan/sarcospan subcomplex, defective in Duchenne muscular dystrophy -cytosolic complex - signaling pathway proteins: nitric oxide synthesis (NOS) - synthesizes gaseous signaling molecule nitric oxide (NO), GRB2 - a component of signaling pathways activated by certain cell-surface receptors

principle types of epithelia

-epithelial cell apical, lateral, and basal surface can exhibit distinctive characteristics (basolateral surface - indistinguishable basal and lateral sides) 1. simple columnar epithelia: elongated cells including mucus-secreting cells (in the lining of the stomach and cervical tract) and absorptive cells (in the lining of the small intestine), microvilli are on the apical surface 2. simple-squamous epithelia: thin cells - including cells lining blood vessels (endothelial cells/endothelium) and many body cavities 3. transitional epithelia: several layers of cells with different shapes - line certain cavities subject to expansion and contraction 4. stratified squamous (nonkeratinized) epithelia: line surfaces such as the mouth and vagina, resists abrasion, generally prevent material absorption/secretion into or out of lines cavity -basal lamina: thin fibrous network of collagen and other ECM components, connects epithelia to underlying connective tissue

onward from the golgi: exocytosis

-exocytosis (secretion): both constitutive (always on) and regulated pathways -constitutive provides cell with newly made lipids and proteins to resupply/replace worn out membrane components as well as increase surface area for cell division -also secretes extracellular proteins that will become a part of the extracellular matrix or diffuse to signal or nourish other cells -proteins being constitutive secreted do not possess a unique signal sequence -proteins whose secretion is regulated are densely aggregated in the trans-golgi network, allowing high concentration of secretion -regulated exocytosis pathways: only occurs in cells specialized fro secretion - such as hormone, digestive, or neurotransmitter producing cells -such secretions are stored in secretory vesicles near the plasma membrane and must wait for a release signal -for example, excess of blood sugar triggers insulin release from pancreatic beta-cells

protein constituents of typical adherens junctions - E-cadherins

-exoplasmic domains - clustered by cis and trans interactions at adherens junctions on adjacent cells, form Ca2+-dependent homophilic interactions -cytosolic domains - bind directly/indirectly through multiple adapter proteins to actin filaments of the cytoskeleton, participate in intracellular signaling pathways

smooth ER

-extensively developed in a number of cell types -functions include: synthesis of steroid hormones in endocrine cells (endocrine cells of the gonad and adrenal cortex), detoxification in the liver of various organic compounds (home of the P450 enzymes), sequestration of calcium ion from cytoplasm of muscle cells (contains a high concentration of calcium-binding protein), synthesis of most membrane lipids (ex: cholesterol)

receptor-mediated endocytosis (RME)

-extracellular molecules bind cell-specific receptor proteins, membrane anchored proteins that bind these ligands with high specificity: uptake of nutrients, metabolites, signaling molecules -receptor binding activates clathrin assembly, concentrates molecule to be absorbed, and allows only cells expressing the receptor to absorb the molecule -receptors in coated pits or move to coated pits -used by cells to import specific macromolecules/complexes too large to be imported by membrane transporters -uptake specificity - receptor-dependent -uptake mechanism - ligand-receptor complexes incorporated into clathrin/AP2-coated vesicles -RME receptors: some types cluster in clathrin-coated pits by cytoplasmic domain association with AP2 even in absence of ligand, other types diffuse freely in the plasma membrane until a ligand-induced conformational change associates them with AP2, two or more types of receptor-bound ligands (such as LDL and transferrin_ can be present in the same coated pit or vesicle

the collagen triple helix

-fibrillar collagens: trimeric proteins made from 3 polypeptides - can be identical (homotrimer) or different (heterotrimer); peptides - encoded by one of at least human 43 collagen alpha chain genes -collagen structure: polypeptide fragment contains repeating sets of Gly-X-Y amino acids characteristic of collagen alpha chains, each chain is twisted into a left-handed helix, triple-helical structure with left-handed twist of the individual collagen alhpa chain, glycine proton side chains

organization of fibronectin and its binding to integrin

-fibronectin (Fn) - a linear array of distinct polypeptides giving it a modular structure -each polypeptide is about 30 Fn modules - found in other proteins too -Fn has binding sites for other components of the ECM -Fn guides migrating cells during embryogenesis -fibronectin connects cells and ECM, influence cell shape, differentiation, and movement -ECM binding - specific binding sites for heparan sulfate, fibrin, and collagen -cell-surface integrin bindings - two type III cell-binding domains - bound by integrin extracellular domains

v-SNARE/t-SNARE complex

-four long alpha helices, two from SNAP-25 and one each from syntaxin (t-SNARE) and VAMP (v-SNARE), form numerous noncovalent interactions to form four-helix coiled-coil -formation of four-helix bundle is energetically favorable and can overcome the electrostatic repulsion of the phospholipid heads -this allows the hydrophobic interiors to come into contact and create an opening between the two membranes; the membranes fuse together and hydrophobic interactions reorder the phospholipids into a bilayer -basically twist-ties

processing of N-linked oligosaccharide chains on glycoproteins within cis, medial, and trans-golgi cisternae in vertebrate cells

-golgi complex - three biochemical processing compartments contain different enzymes that modify proteins post-translationally -anterograde transport through the three golgi processing compartments occurs by cisternal maturation -glycosylation in the golgi complex: sequence of incorporation of sugars into oligosaccharides is determined by glycosyltransferases in each region of the golgi; glycosylations steps can be diverse

onward to the golgi complex

-golgi complex is a stack of flattened membrane cisternae, some cisterna continuous with others -golgi complex receives COP coated vesicles from the ER and further modifies proteins in transit and sorts them for delivery -many golgi per cell, number depends on degree of protein production -cis-golgi network (CGN): entry site for material arriving from ER; some proteins separated and packaged into vesicles then recycles to the ER, other proteins sent on to other regions of the golgi -trans-golgi: delivery -cisternae: modification

phagocytosis

-in multicellular organisms, phagocytosis is used infrequently for nutrient uptake (this is primarily accomplished by transporters and channels specific to certain molecules) -rather, in multi-tissue organisms phagocytosis is restricted to cells of the immune system (white blood cells like macrophages and neutrophils) that fight invasion by foreign microorganisms -used in single-cell protozoans as a feeding mechanism -cell surface receptors recognize the foreign bodies either by the presence of specific molecules or because they have been tagged by host antibodies -mediated by arrangement of cytoskeleton -polymerization of the cytoskeleton generates cytoplasmic extensions called pseudopods that engulf the foreign body -coat-independent process: fusion of membrane forms a phagosome that delivers the invader ultimately to lysosomes for degredation

mechanism of nuclear import

-in the nucleus, importin/cargo complex binds a GTPase names Ran (which is activated by guanine exchange factors (GEF) protein) -Ran-GTP binds importin causing it to dissociate from the cargo -Ran-GTP/importin is bounds by an export protein and transported through the pore -in the cytoplasm, another protein (GTPase activating proteins) activate the RAN GTPase activity causing it to hydrolyze GTP -importin is then released from the Ran-GDP molecule and is able to bind new cargo -it is the hydrolysis of GTP that drives this process by maintaining a high concentration of importin in the cytoplasm -nucleoplasm (GTP-bound): high Ran-GTP and GEF -cytosol (GDP-bound): high Ran-GDP and GAP

principal types of cell junctions connecting the columnar epithelial cells lining the small intestine

-individual cells can form multiple types of junctions -basal surface - adhesion to basal lamina -apical surface - fingerlike microvilli project into the intestinal lumen -tight junction: primarily in epithelial cells, surrounds the cell below the microvilli - connects to all neighboring cells, regulates paracellular transport of substances between the intestinal lumen and internal body fluids (blood) via the extracellular space between cells, boundary between apical and basolateral regions of the plasma membrane -gap junctions: allow movement of small molecules and ions between cytosols of adjacent cells -adhesion junctions: adherens junction (continuous junction with all neighboring cells, circumferential belt of actin and myosin filaments associated with the adherens junction - functions as tension cable that can internally brace and control cell shape), desmosomes (spot cell-cell junctions, utilize cadherin proteins for cell to cell contact), hemidesmosomes (spot cell-ECM junctions, utilize integrin proteins for cell to ECM contact)

interactions of cells with extracellular materials

-integrins - family of membrane proteins composed of heterodimers with a and b subunits -have major role in integrating extracellular and intracellular environments -another role is adhesion of cells to their substratum or other cells -binding to ECM is calcium-dependent -found only in animal cells

integrins couple the ECM with the cytoskeleton

-integrins join cells to the ECM through association with fibronectin and these other linker proteins -over 24 human integrins that bind distinct extracellular protein -each also has unique cytoplasmic domains that in addition to interacting with the cell cytoskeleton, transduce extracellular signals -multiple integrin proteins can be found in the same cell type -binding an integrin to its cognate extracellular molecule causes a conformational change that promotes activation of the cytoplasmic domain -likewise, intracellular signaling molecules may bind integrin and promote an active conformation that promotes binding to extracellular molecules -these interactions are important not only for adhering to extracellular surfaces, but relay signals for cell survival

integrin adhesion receptor-mediated signaling pathways control diverse cell functions

-interaction via adapter proteins and signaling molecules with a broad array of intracellular signaling pathways -influence cell survival, gene transcription, cytoskeletal organization, cell motility, and cell proliferation -inside-out signaling - intracellular signaling pathways cause adapter proteins to modify integrin ability to bind extracellular ligands (usually components of the ECM) -outside-in signaling - extracellular ligand binding changes integrin cytoplasmic domain conformation; alters interactions with cytoplasmic proteins including adapter proteins and signaling kinases that transmit signal via diverse signaling pathways; influences cell proliferation, cell survival, cytoskeletal organization, cell migration, and gene transcription; components of several signaling pathways are associated directly with the plasma membrane (some) or shared with other cell-surfce activated signaling pathways (most)

endothelium-leukocyte interactions: activation, binding, rolling, and extravasation

-leukocyte extravasation through a blood vessel wall into tissues - orchestrated by a precisely timed sequence of adhesive interactions -4 types of leukocytes extravasate into tissues: 1. neutrophils - release several antibacterial proteins 2. monocytes - precursors of macrophages that phagocytose and destroy foreign particles 3 and 4. T and B lymphocytes - antigen-recognizing cells of the immune system

models of domains in mechanosensor proteins responding to mechanical forces

-mechanotransduction: reciprocal interconversion of a mechanical force/stimulus and biochemical processes -extracellular mechanosensors - respond to the mechanical stimulus by changing shape and activity 1. fibronectin type III domain unfolds when subjected to mechanical force: mechanical force generated within the cell by cytoskeleton alterations and mechanotransduced via multiple integrin adhesion receptors bound to the extracellular matrix, fibronection domain unfolding exposed a putative previously hidden binding site - potential to form beta sheets with other fibronectin molecules to form fibronectin fibrils for ECM assembly 2. talin (intracellular integrin adapter protein) domain partial unfolding under mechanical stretch

delivery of plasma-membrane proteins to the lysosomes interior for degredation

-membrane proteins targeted for degradation - delivered to the lysosome lumen -step 1 - vesicles carrying newly synthesized lysosomal membrane proteins from the trans-golgi network fuse with the late endosome -step 2 - endosomes carrying endocytosed plasma-membrane proteins targeted for degradation - fuse with the late endosome -step 3 - late endosome - plasma membrane proteins targeted for degradation - incorporated into vesicles that bud into the interior of the late endosome, forms a multivesicular endosome -step 4 - multivesicular endosome fuses with a lysosomes - internal vesicles degraded, lysosomal membrane proteins - not degraded

trafficking of soluble lysosomal enzymes from the trans-golgi network and cell surface to lysosomes

-missorted M6P enzymes: imperfectly sorted enxymes - constitutively secreted (could cause extracellular damage) -step 6: plasma membrane M6P receptors capture missorted M6P-enzymes into calthrin-coated vesicles in the receptor-mediated endocytosis pathway

protein import

-mitochondrial and chloroplast proteins - imported and localized in multiple organelle compartments by several mechanisms -nuclear proteins - enter and exit through pores in the nuclear envelope

review

-mitochondrial and chloroplast proteins encoded by nuclear genes are synthesized on cytosolic ribosomes, and maintained in an unfolded state by chaperones -N-terminal targeting sequences direct post-translational transport of the unfolded proteins through translocons into the organelles -multiple internal targeting sequences direct protein targeting inside the organelles to different membrane and lumen destinations

model of the mechanism for formation of multivesicular endosomes

-most proteins targeted for a multivesicular endosome degradation - tagged with ubiquitin at the plasma membrane, in the trans-golgi network, or inn the endosomal membrane -multivesicular endosome formation mechanism: ubiquinylatedd protein on the endosomal membrane directs loading of ubiquitinylated membrane cargo proteins into endosome vesicle buds - recruits and assembles cytosolic ESCRT protein complexes on the endosome membrane -ESCRT complexes - mediate the completion and pinching off of inwardly budding vesicles -hydrolysis - drives disassembly/recycling of ESCRT complex proteins

proteins enter the nucleus through nuclear pores

-nuclear envelope is composed of two concentric acid and continuous membranes -inner and outer membranes have distinct protein complements -inner membrane possesses proteins that anchor chromosomes and associate with the nuclear lamina: a meshwork of structurally supporting proteins -outer nuclear envelope is very similar in comparison to ER

the nuclear pore complex

-nuclear pores are large protein complexes that span both inner and outer membrane and provide regulated passage of specific molecules into and out of nucleus -major structural features - formed by membrane, structural, and FG-nucleoporins -FG-nucleoporins 1. extended disordered structures composed of Phe-Gly sequence repeats interspersed with hydrophilic regions 2. FG-repeat sequences - fill the central channel with a gel-like matrix 3. gel-like matrix - bulk flow properties that allow diffusion of small molecules but block unchaperoned translocation of proteins larger than 40 kDa -like mesh in a sink strainer -import/export of proteins, RNAs, and ribosomal subunits )which are synthesized in the nucleus) is regulated by proteins possessing nuclear localization signals (NLS) and nuclear export signal (NES)

targeting sequences in imported mitochondrial proteins

-nuclear-encoded mitochondrial proteins - N-terminal matrix-targeting sequence -proteins destined for the inner membrane, the intermembrane space, or the outer membrane - have one or more additional targeting sequences -three pathways - all involve Tom40 to cross the outer membrane

Ran-independent nuclear export

-nucleoplasm: heterodimeric NXF1/NXT1 nuclear export receptor complex binds to mRNA-protein complexes (mRNPs); complex diffuses trhough NPS by transiently interactins with FG nucleoporins -cytoplasm: and RNA helicase (Dbp5) located on the cytoplasmic side of the NPC uses ATP energy to remove NXF1 and NXT1 from the mRNA -recycling system: the Ran-dependent import process recycles free NXF1 and NXT1 proteins back into the nucleus; only goes forward

the unfolded protein response

-occurs when quality control system is overwhelemd: misfolded proteins accumulate in ER, for example under certain stresses -presence of unfolded proteins in the rough ER increased transcription off genes that encode ER chaperones and other folding catalysts -Ire1 - ER membrane transmembrane, luminal domain - binds excess BiP (not associated with unfolded proteins), cytosolic domain - specific RNA endonuclease -step 1 - accumulating unfolded proteins in the ER lumen bind available BiPs -step 2 - Ire1 endonuclease activity in the cytosol cuts an unspliced mRNA precursor encoding the transcription factor Hac1 -step 3 - the two Hac1 exons join to form a functional Hac1 mRNA -step 4 - Hac1 is translated into Hac1 protein, which moves into the nucleus and activates transcription of genes encoding several protein-folding chaperones -if UPR cannot correct the folding error, the UPR triggers activation of the apoptotic pathway - programmed cell death

addition and initial processing of N-linked oligosaccharides

-oligosaccharide chains promote protein folding, stability, adhesion, and recognition -step 1 - Glc3Man9 precursor -step 2-4 - glucose residues removed -step 3 - glucose residue removed, the re-addition of one glucose residue

3 pathways to the inner mitochondrial membrane from the cytosol

-path A: N-terminal matrix-targeting sequence recognized by Tom import receptor in the outer membrane; hydrophobic stop-transfer anchor sequence - blocks transfer through TIM; released from TIM into inner membrane -path B: ATP synthase subunit 9 - matrix-targeting sequence and internal hydrophobic domains recognized by the Oxa1 inner-membrane protein; N-terminal matrix-targeting sequence; Oxa1 inserts protein into inner membrane -path C: ATP/ADP antiporter - lack N-terminus atrix-targeting sequence

endocytosis

-pinocytosis: aka bulk-phase endocytosis, non-specific uptake of cellular fluids, used for retrieval of membrane components -receptor-mediated endocytosis: uptake of specific molecules, bind to receptors in cell membrane - transmembrane proteins, bindings sit on exterior of cell, different receptors for different ligands -specialized cells are able to internalize large particle, even other cells -calthrin-coated pits generate endocytic vesicles that are delivered to endosomes -ingested material can be recycled or sent to lysosomes -pinocytosis and receptor-mediated endocytosis are clathrin-dependent -phagocytosis: non clathrin dependent, cells and cell debris, larger vesicles called phagosomes

the dynamics of transport through the golgi - cisternal maturation model

-polarity of the golgi: 1. cis face of golgi closest to ER - network of tubules (CGN) 2. trans face of golgi away from ER - network of tubules (TGN) -cisterna formed by fusion of vesicles at cis face; moves to the next position as new cisterna formed; progresses down golgi stack -discrete enzymatic activity in each region of the golgi and these enzymes are moved in an anterograde fashion along with the proteins being processed -retrograde movement of transport vesicles (COP coated) returns these enzymes to their appropriate positions both in the golgi and the ER -evidence: 1. blocking ER transport vesicles leads to disappearance of golgi 2. materials produced in ER remain in the golgi complex and never appear within the golgi-associated transport vesicles 3. data suggests that vesicles can move forwards or backward 4. composition of individual golgi cisterna change over time -movement of material: CGN to cis cisterna to medial cisternae to trans cisterna to TGN -membranous elements of golgi complex supported mechanically by a peripheral membrane scaffold -scaffold physically linked to motor proteins that direct movement of vesicles -golgi 'matrix' is a group of fibrous proteins that play a key role in the disassembly and reassembly of the golgi complex during cell division

hydropathy profiles

-predicts likely topogenic sequences in integral membrane proteins -plot of total hydrophobicity of each 20 contiguous amino acid segment along the length of a protein -results: (+) values - relatively hydrophobic regions of the protein, (-) values - relatively hydrophilic regions of the protein -complex profiles for multipass (type IV) proteins often must be supplemented with other analyses to determine topology

signal sequences direct proteins to correct compartment

-primary sequence of many proteins contains specific signal sequence: a sorting signal composed of a unique amino acid sequence that is recognized by other cellular proteins and used as a mailing address for delivery or retention -typically a continuous stretch of 4-60 amino acids -often but not always cleaved from the mature protein once it has reached its destination -organelle targeting sequences are similar in their location and general function but differ in sequence characteristics: ER - sequence on N-terminus, is eventually removed, and is a core of 6-12 hydrophobic amino acids often preceded by one or more basic amino acids

proteins that compose tight junctions - occludin and claudin

-principle integral membrane proteins of tight junctions -four transmembrane domains -occludin C-terminal cytoplasmic domain binds PDZ-containing scaffold proteins -claudin larger extracellular loop - contributes to paracellular ion transport selectivty -JAM protein - single transmembrane domain, extracellular domain (two immunoglobulin domains)

retaining and retrieving resident ER proteins

-proteins are maintained in an organelle by a combination of two mechanisms: retention of resident molecules that are excluded from transparent vesicles and retrieval of 'escaped' molecules back to the compartment in which they reside -proteins that normally reside in ER contain short amino acid sequences at their C-terminus that serve as retrieval signals -specific receptors capture the molecules and return them to the ER in COP1 coated vesicles: KDEL receptor - recognizes and returns soluble ER proteins based on KDEL signal; ER membrane proteins have a KKXX retrieval signal on their C-terminus which binds to COPI coat

action of the nuclear pore complex

-proteins with an NLS are recognized by a cytosolic nuclear import receptor complex commonly called importin -the importing/cargo complex binds fibrils on the outer rim of the pore and is drawn into the meshwork of fibrils in its center, thus allowing diffusion into the nucleus -nuclear transport is however and active process that requires hydrolysis of GTP

Ran-dependent nuclear export

-proteins, tRNAs, and ribosomal subunits are exported form the nucleus to the cytoplasm -nuclear-export signal (NES) - leucine-rich region -nucleoplasm: exportin 1 - binds cooperatively with and NES-cargo protein and Ran-GTP; complex diffuses through and NPC vis transient interactions with FG-repeats in FG-nucleoporins -cytoplasm: Ran-GAP assocaited with the NPC cytoplasmic filaments stimulates Ran-GTP hydrolysis to Ran-GDP; Ran-GDP conformational change releases NES-containing cargo protein into the cytosol -recycling system: exportin 1 and Ran-GDP - transported back into the nucleus; Ran-GEF in the nucleoplasm converts Ran-GDP to Ran-GTP

extracellular matrix proteins

-proteoglycans - a unique type of glycoprotein -collagens - form fibers -fibronectin and laminin - multi-adhesive matrix proteins - important organizers of the extracellular matrix; king, flexible molecules that contain multiple domains; bind various types of collagen, other matrix proteins, polysaccharides, and extracellular signaling molecules as well as adhesion receptors; interactions with adhesion receptors - regulate cell-matrix adhesion and cell shape behavior

the repeating disaccharides of glycosaminoglycans (GAGs)

-proteoglycans and constituent GAGs play diverse roles in the ECM -four classes of GAGs: formed by polymerization of monomeric units into repeats of a particular disaccharide and subsequent modifications -modifications: addition of sulfate groups, inversion of the carboxyl group, heparain - hypersulfation of heparan sulfate, hyaluronan - nonsulfated

variation in the relative density of cells and ECM in different tissues

-relative volumes occupied by cells and surrounding matrix vary greatly among different animal tissues -dense connective tissue - contains mostly extracellular matrix containing tightly packed ECM fibers interspersed with rows of relatively sparse fibroblasts (cells that synthesized ECM) -sparse connective tissue - squamous epithelial cells tightly packed into a quilt-like pattern with little ECM between the cells

vesicle-mediated protein trafficking between the ER and cis-golgi

-reverse (retrograde) transport - COPI vesicle-mediated cis-golgi to ER transport -cargo - recycles the membrane bilayer, v-SNAREs, and missorted ER-resident proteins

classes of ER membrane proteins

-rough Er synthesizes five topological classes of integral membrane proteins and a sith type tethered to the membrane by a phospholipid anchor -integral membrane proteins: classified by orientation in the membrane, locations of N- and C-termini, and the types of targeting signals they contain; hydrophobic alpha-helices segments - embed in the membrane bilayer; hydrophilic regions - fold into various conformations outside the membrane -type IV proteins - multiple transmembrane alpha helices (multipass membrane proteins)

focal adhesions

-scattered, discrete sites for cell adhesion to their substratum in vitro -may act as a type of sensory structure to facilitate cell growth -are also implicated in cell locomotion -used for transient contact to ECM during cell locomotion -integrins mediate adhesion and relay signals between cells and their 3D environments -integrin-based adhesion shape, distribution, and composition varies depending on cell environment -other adhesions include fibrilar adhesions and podosomes

overview of the secretory and endocytic pathways

-secretory and endocytic pathways protein trafficking - unifying principle: transport vesicles transport membrane and soluble proteins from one membrane-bounded compartment to another -transport vesicles: collect cargo proteins in membrane budding from a donor compartment, deliver cargo proteins to the next compartment by fusing with the target membrane -secretory pathways: distribution of soluble and membrane proteins synthesized by the rough ER to final destinations at the cell surface (including secretion) or in lysosomes -two stages of secretory pathways: stage 1 - rough ER 1. synthesis of proteins bearing an ER signal/targeting sequence - cotranslational insertion of newly made polypeptide chains into the ER membrane or across it into the ER lumen stage 2 - protein trafficking 2. proteins packaged into transport vesicles that bud from the ER and fuse together to form a new cis-Golgi cisternae 3. ER enzymes or structural proteins - retained in the ER and retrieved to the ER by vesicles that bud from the cis-golgi and fuse with the ER 4. each cis-golgi cisterna and contents moves from the cis to the trans face of the golgi complex by nonvesicular cisternal maturation 5. retrograde transport vesicles move golgi-resident proteins to the previous golgi compartment 6. constitutive secretions (all cells) - transport vesicles move continuously and fuse with the plasma membrane; soluble proteins are continuously secreted, membrane proteins become plasma membrane proteins

sorting of proteins destined for the apical and basolateral plasma membranes of polarized cells

-several pathways sort membrane proteins to apical or basolateral membrane regions of polarized cells with tight junctions -some cellular proteins - sorted similarly to only apical (including GPI proteins in some cell types) or basolateral membranes -some hepatocyte apical membrane proteins: sorted initially to basolateral membrane, selectively endocytosed into clathrin-caoted vesicles and transcytosed through endosomes to the apical membrane

structure and assembly of type IV collagen

-sheet-forming type IV collagen is a major structural component of the basal lamina -N-terminus - small globular domain, C-terminus - large globular domain -collagenous triple helix - interrupted by nonhelical segments that introduce flexible kinks into the molecule -formation of dimers, tetramers, and higher-order sheet-like networks: lateral interactions between triple-helical segments, head-to-head and tail-to-tail interactions between the globular domains, multiple covalent cross-links between lysine and methionine amino acids and some adjacent C-terminal domains and contribute to the stability of the network

gap junctions: mediating intercellular communication

-sites between animal cells for intercellular communication -composed entirely of membrane protein connexin - 21 different connexin genes - different sets of connexins are expressed in different cell types -connexins are organized into a complex called connexon - composed of 6 individual connexon proteins -allow small molecules to pass directly between cytosols of adjacent cells -gap-junction intercellular communication (GJIC) allows the passage of low-weight molecules -gap junctions can allow integration of activities of individual cells into a functional unit -compatibility differences between connexins either promote or prevent communication between different cells -regulated by post-translational modification of connexins -sensitive to changes in environmental conditions such as intracellular pH and calcium concentration, membrane potential, and intercellular potential between adjacent interconnected cells (voltage gating)

lysosomes

-small to relatively large -spherical to irregular in shape -bounded by single membrane -heterogeneous interior -functions: 1. organelles for cellular digestion in animal cells -acid hydrolases for hydrolysis of almost every type of biological macromolecule (pH optimum in acid range) 2. degradation of ligands and dissolved macromolecules taken up by endocytosis 3. digestion of solid materials brought into cells by phagocytosis - phagolysosome formed by fusion of lysosomes with phagosome 4. digestion of cellular organelles by autophagy (self-eating): organelles surrounded by membrane from ER, fusion of autophagic vacuole with lysosomes; purpose of autophagy - turnover of organelles during differentiation, destruction of damaged organelles, digestion of organelles during starvation

proteolytic processing of proproteins in the constitutive and regulated secretory pathways

-some proteins undergo proteolytic processing after leaving the trans-golgi -proproteins: matured into final form after leaving the trans-golgi, include soluble lysosomal enzymes, membrane proteins such as influenza hemaglutinin (HA), and secreted proteins such as serum albumin, insulin, glucagon, and the yeast alpha mating factor

post-translational translocation

-some yeast secretory proteins enter the ER lumen through the Sec61 translocon after translation has been completed (no SRP/receptor involvement) -step 1 - direct interaction between the SS and the translocon (sufficient for targeting to the ER membrane), N-terminal segment of the protein enters the ER lumen (signal peptidase cleaves the signal sequence just as in cotranslational translocation) -step 2 - BiP (Hsp70 family protein of ATP-dependent molecular chaperones) and Sec63 complex - provide driving force for unidirectional translocation across the ER membrane -BiP interaction with Sec63 complex - stimulates BiP ATP hydrolysis -BiP-ADP conformational change - promotes binding to exposed polypeptide chain translocating through adjacent translocon -BiP binding prevents backsliding of peptide through translocon

tight junctions

-specialized contacts between epithelial cells -seal off body cavities -located at very apical end of the junctional complex between adjacent cells -serve as barrier to free diffusion of water and solutes from the extracellular compartment -some are permeable to specific ions or solutes - permeability varies on composition of proteins

left secretory pathway

-step 1 - ribosomes initiate nascent protein synthesis in the cytosol -step 2 - ER signal sequence directs ribosome docking onto rough ER import apparatus, nascent proteins translocated into the ER lumen or embedded in the ER membrane -step 3 - proteins in ER membrane or lumen can move via transport vesicles to the golgi complex -step 4a or 4b - further sorting of proteins to the plasma membrane or to lysosomes in vesicles

right nonsecretory pathways

-step 1 - synthesis of proteins lacking an ER signal (targeting) sequence is completed on free ribosomes -step 2 - proteins that contain no targeting sequence remain in the cytosol - steps 3-6 - proteins with an organelle-specific targeting sequence - imported from the cytosol into mitochondria, chloroplasts, peroxisomes, or the nucleus

formation of mannose 6-phosphate (M6P) residues that target soluble enzymes to lysosomes

-sugar residues added post-translationally to proteins in the golgi complex can serve as an 'address label' to target proteins to the proper vesicles for delivery from the golgi complex -golgi enzymes recognize specific amino acids within the protein sequence -M6P targeting signal addition by two cis-golgi-residnet enzymes ultimately leaving a 6-phosphorylated mannose residue on the lysosomal enzyme -M6P residues direct newly synthesized lysosomal enzymes to lysosomes -golgi complex is the mail center

other functions associated with the golgi complex

-synthesis and modification: 1. sphingomyelin synthesis completed 2. O-linked oligosaccharide added to proteins - attached to oxygen of serine or threonine 3. addition of oligosaccharides to lipids 4. site of synthesis of most of cell's complex polysaccharides - polysaccharides of extracellular matrix of animal cells and of cell walls of plants except cellulose

coated vesicles involved in protein trafficking

-three major types of transport-specific coated vesicles: each with a different type of protein coat formed by reversible polymerization of a distinct set of coat and G protein subunits -a conserved set of monomeric GTPase switch proteins controls the assembly of different vesicle coats -clathrin - protein coat - ARF -COPII - Sar1 -COP1 - ARF -COPII-coated vesicles - move materials from the ER to the golgi complex (anterograde motion - away from nucleus) -COPI-coated vesicles - move materials from golgo 'backward' to ER, or from the trans golgi to the cis golgi cisternae (retrograde motion - towards nucleus) -clathrin-coated vesicles - move materials from the TGN to endosomes, lysosomes, and plant vacuoles

overview of vesicle budding and fusion with a target membrane

-three types of coated vesicles mediate protein transport through different pathways -small GTPase proteins direct coat protein polymerization on donor membranes to pinch off vesicles carrying different cargoes -coat shedding exposed Rab and SNARE proteins that target vesicles for fusion with specific target membranes -vesicles: bud from the donor membrane, fuse with specific target membrane, assembly of a protein coat drives vesicle formation and selection of specific cargo molecules -vesicle budding from donor membrane: donor membrane has specific SNARE proteins (vesicle-SNAREs; v-SNAREs) captured in budding vesicle membrane; donor membrane fusion pinches off coated vesicle; coated vesicle uncoated in cytosol - uncovers projecting v-SNAREs for fusion targeting

transcellular and paracellular pathways of transepithelial transport

-tight junctions block transport of many nutrients across the intestinal epithelium between cells through the paracellular pathways -paracellular transport through tight junctions: molecules move extracellularly through tight junctions; tight junction permeability to small molecules and ions depends on composition of the junctional components and the physiological state of the epithelial cell; permeability to ions, small molecules, and water varies enormously among different epithelial tissues -transcellular transport of molecules across epithelia - cellular uptake on one side and subsequent release on the opposite side

membrane insertion and orientation of type 1 single-pass transmembrane proteins

-topology: N-terminus in ER lumen, single transmembrane domain, C-terminus in cytosol -step 1 - translocation initiation and SS cleavage by the same mechanism as for soluble secretory proteins -step 2 - nascent peptide elongates -step 3 - elongation continues until a hydrophobic stop-transfer anchor sequence enters the translocon - prevents nascent chain from extruding farther into the ER lumen

chaperones and other ER proteins (including carbohydrate-binding lectins) facilitate rapid folding and assembly of nascent protein

-transient BiP (chaperone) binding to nascent proteins regions as it enters ER - helps prevent premature folding into incorrect conformation -calnexin and calreticulin (lectins) bind to certain oligosaccharide chains (seven N-linked oligosaccharide chains) - helps prevent premature folding into incorrect conformation -PDI catalyzes formation of disulfide bonds; properly folded monomer folding chaperones

cotranslational translocation

-translation and translocation occur simultaneously -step 1 - N-terminal ER signal sequence (SS) emerges from the ribosome first during nascent protein synthesis -step 2 - signal recognition particle (SRP) binds SS - arrests protein synthesis -step 3 - SRP-nascent polypeptide chain-ribosome complex: binds to the SRP receptor in the ER membrane, interaction is strengthened by the binding of GTP to both the SRP and its receptor -step 4 - transfer of the nascent polypeptide-ribosome to the translocon: opens translocation channel to admit the growing polypeptide, SS transferred to a hydrophobic binding site next to the central pore, SRP and SRP receptor hydrolyze bound GTP, dissociates SRP from ribosome and receptor, restarts protein synthesis (can initiate the insertion of another polypeptide chain) -step 5 - elongating polypeptide chain: passes through the translocon channel into the ER lumen, SS cleaved by signal peptidase and rapidly degraded -step 6 - growing peptide chain - continues extrusion through the translocon into the ER as the mRNA is translated toward the 3' end -step 7 - translation completes at mRNA stop codon- ribosome is released -step 8 - nascent protein (remainder is drawn into the ER lumen and folds into native conformation) and translocon closes

structure of an archaeal Sec61 complex

-translocon - preserves integrity of the ER membrane by two gating mechanisms -hourglass-shaped channel through the center of the pore: (-) peptide - channel closed by a short helical plug - moves out of the channel during protein translocation (one gating mechanism); (+) peptide - ring of isoleucine residues at the constricted wast of the pore - forms a gasket that keeps the channel sealed to small molecules even when translocating a polypeptide (second gating mechanism) -channel helices - may separate for lteral passage of a nascent protein hydrophobic transmembrane domain into the ER membrane lipid bilayer

interactions of fibrillar collagens with fibril-associated collagens

-type I and II collagens - associate with nonfibrillar collagens to form diverse structures, ex: tendons and cartilage

topogenic sequences determine the orientation of ER membrane proteins

-type I proteins - N-terminal signal sequence (cleaved) + signal internal stop-transfer anchor) STA-sequence -type II - single internal signal-anchor (SA) sequence, orientation depends on a high density of positively charged amino acids (+++) on the N-terminal side of the SA sequence -type III - single internal signal-anchor (SA) sequence, orientation depends on a high density of positively charged amino acids (+++) on the C-terminal side of the SA sequence -type IV-A proteins - no N-terminal SS, N-terminus in the cytosol, alternating type II SA sequence and STA sequences -type IV-B proteins - no N-terminal SS, N-terminus in the ER lumen, type III SA sequence followed by alternating type II SA and STA sequences

membrane insertion and orientation of type II and III single-pass transmembrane proteins

-type II and III proteins: lack N-terminal SS, have a single internal hydrophobic signal-anchor sequence - both ER signal sequence and membrane anchor -type II: 1. SRP binds to the internal signal-anchor sequence synthesized by a cytosolic ribosome and interacts with the SRP receptor on the ER membrane (positively charged amino acids on N-terminal side of signal-anchor sequence orient nascent polypeptide chain in the translocon with the N-terminal portion in the cytosol 2. chain elongation extrudes remainder of nascent protein into the ER lumen 3. internal signal-anchor sequence - moves laterally through a hydrophobic cleft between translocon subunits to anchor the protein in the ER phospholipid bilayer (completion of protein synthesis - releases ribosome subunits into cytosol and peptide C-terminus into the ER lumen -type III: 1. insertion similar to that of type II proteins - except positively charged residues on the signal-anchor sequence C-terminal side cause transmembrane segment orientation within the translocon with C-terminal end in the cytosol and the N-terminal end in the ER lumen 2. C-terminal elongation is completed in the cytosol 3. ribosomal subunits are released -type II and III proteins flip orientation if signal-anchor sequence-flanking positively charged residues are mutated

two types of receptors subjected to endocytosis

1. house-keeping receptors - responsible for uptake of materials that will be used by the cell: transferrin (mediates iron uptake), LDL receptors (mediate cholesterol uptake), generally lead to delivery of the bound material to the cell and the return of the receptor to the plasma membrane 2. signaling receptors - responsible for binding ligands that carry messages that change the activities of the cell: hormones, growth factors, receptor-down regulation is the internalization of signaling receptors that usually leads to degradation of the receptor after signaling cascade is initiated

most cellular proteins synthesized in 1 of 2 places

1. in cytoplasm of 'free' ribosomes not bound to membranes -proteins in cytosol -peripheral proteins on membranes -proteins transported into nucleus -proteins incorporated into peroxisomes, mitochondria, and plastids 2. bound to surface of RER -secreted proteins -soluble and integral membrane proteins of ER, golgi complex, lysosomes, endosomes, vesicles, and plant vacuoles

model of peroxisomal biogenesis and division

de novo formation of peroxisomes: -first stage: incorporation of peroxisomal membrane proteins into precursor membranes derived from the ER -peroxisomal membrane protein insertion - produces a peroxisomal ghost, which imports matrix-targeted proteins -proliferation - most peroxisomes arise from Pex11-dependent division of mature peroxisomes

protein import to organelles

delivery of proteins and lipids to organelles is required for: -organelle-specific function -growth and replication of organelles -and therefore cell division as each daughter cell requires a complement of these structures protein sorting is a complex delivery system that utilizes a combination of three processes and always begins with initiation of protein translation on ribosomes in the cytoplasm with the exception of some proteins produced directly in the mitochondria or chloroplasts

necessity and sufficiency of signal sequences

import mechanism experiment: -cell-free system synthesis of mitochondrial precursor proteins containing uptake-targeting sequences -addition of respiring mitochondria to presynthesized mitochondrial precursor proteins: proteins taken up by mitochondria - protected from added proteases -control - proteins degraded when no mitochondria added -trypsin digests proteins not in the mitochondria

two pathways to the mitochondrial intermembrane space

path A: cytochrome b2 -N-terminal matrix-targeting sequence - removed by the matrix protease -stop transfer sequence: blocks translocation of the protein across the inner membrane; causes release from TIM into the inner membrane (similar to inner membrane protein path A) -inner membrane protease - cleaves protein on the intermembrane side of the stop-transfer sequence path B: specialized pathway for delivery of the intermembrane chaperones Tim9 and Tim10 -no N-terminal matrix-targeting sequence or tim targeting sequence

PTS1-directed import of peroxisomal matrix proteins

peroxisome luminal proteins: -all encoded by nuclear genes -synthesized on free ribosomes in the cytosol -incorporated into preexisting or newly generated peroxisomes -C-terminal peroxisomal-targeting sequeny PTS1 -PTS1 recognized by cytosolic receptor - targets proteins for transport to peroxisome lumen mechanism 1. PTS1 targeting sequence bind to the cytosolic receptor Pex5 2. Pex5-matrix protein complex forms multimeric complex with the Pex14 receptor in the peroxisomal membrane 3. matrix protein dissociates from Pex5 and s released into the peroxisomal matrix 4-5. Pex5 returns to the cytosol - RECYCLED