Ch 13

Transient BiP

(chaperone) binding to nascent proteins regions as it enters ER - helps prevents premature folding into incorrect conformation

rough ER

-Composed of a network of flattened sacs (cistenae). -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.

signal-recognition particle

-ER signal sequence on N-terminus is bound by a cytoplasmic protein called the_____________ _________________ _________________(SRP). -delivers the ribosome/mRNA/peptide complex to the SRP receptor on the membrane of the RER

nonsecretory pathways

-Synthesis of proteins lacking an ER signal (targeting) sequence is completed on free ribosomes. -Proteins that contain no targeting sequence remain in the cytosol. -Proteins with an organelle-specific targeting sequence (pink) - imported from the cytosol into mitochondria, chloroplasts, peroxisomes, or the nucleus

Endoplasmic Reticulum (ER)

-comprises a network of membranes that penetrates much of the cytoplasm.

secretory pathway

1. Ribosomes initiates nascent protein synthesis in the cytosol. 2. ER signal sequence (pink) directs ribosome docking onto rough endoplasmic reticulum (ER) import apparatus. •Nascent proteins translocated into the ER lumen or embedded in the ER membrane. 3. Proteins in ER membrane or lumen can move via transport vesicles to the Golgi complex. 4. Further sorting of proteins to the plasma membrane or to lysosomes in vesicles

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; often but not always cleaved from the mature protein once it has reached its destination

mitochondrial matrix

Amphipathic N-terminal targeting sequences target proteins to the _________________ ___________.

polyribosome

Both free and bound ribosomes translate mRNA in _______________ complexes: a single mRNA bound by multiple ribosomes at various stages of translation

cytosolic and ER destined proteins

Common pool of ribosomes synthesizes both ____________ and __________ destined proteins

•Organelle-specific function •Growth and replication of organelles •And therefore cell division as each daughter cell requires a complement of these structures.

Delivery of proteins and lipids to organelles is required for:

SRP binds the internal signal-anchor sequence synthesized by a cytosolic ribosome (not shown) and interacts with the SRP receptor on the ER membrane.

Explain step 1 of Membrane insertion and orientation of type II

Insertion similar to that of type II proteins

Explain step 1 of Membrane insertion and orientation of type III

•N-terminal ER signal sequence (SS) emerges from the ribosome first during nascent protein synthesis.

Explain step 1 of cotranslational translocation.

complex sequesters the nascent protein hydrophobic C-terminal tail anchor sequence (not shown) and transfers it to Get3-ATP

Explain step 1 of insertion of tail-anchored proteins (C-terminal)

Translocation initiation and signal sequence cleavage by the same mechanism as for soluble secretory proteins

Explain step 1 of membrane insertion and orientation of type 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)

Explain step 1 post-translational translocation.

•Chain elongation extrudes remainder of nascent protein into the ER lumen.

Explain step 2 of Membrane insertion and orientation of type II

•C-terminal elongation is completed in the cytosol.

Explain step 2 of Membrane insertion and orientation of type III

•Signal recognition particle (SRP) binds SS - arrests protein synthesis

Explain step 2 of cotranslational translocation.

•Get3-ATP-nascent protein complex docks onto the ER membrane dimeric Get1/Get2 receptor.

Explain step 2 of insertion of tail-anchored proteins (C-terminal)

•Nascent peptide elongates

Explain step 2 of membrane insertion and orientation of type 1

•BiP (Hsp70 family protein of ATP-dependent molecular chaperones) and Sec63 complex - provide driving force for unidirectional translocation across the ER membrane

Explain step 2 post-translational translocation.

Ribosomal subunits are released.

Explain step 3 of Membrane insertion and orientation of type III

•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.

Explain step 3 of cotranslational translocation

•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.

Explain step 3 of insertion of tail-anchored proteins (C-terminal)

Elongation continues until a hydrophobic stop-transfer anchor sequence enters the translocon - prevents nascent chain from extruding farther into the ER lumen

Explain step 3 of membrane insertion and orientation of type 1

•Random inward sliding - exposes more nascent protein

Explain step 3 post-translational translocation.

•Transfer of the nascent polypeptide-ribosome to the translocon - •Opens translocation channel to admit the growing polypeptide •Signal sequence - 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)

Explain step 4 of cotranslational translocation

Get3 release of ADP and binding ATP releases it from Get1/Get2

Explain step 4 of insertion of tail-anchored proteins (C-terminal)

Stop-transfer anchor sequence moves laterally through a hydrophobic cleft between translocon subunits and becomes anchored in the phospholipid bilayer.

Explain step 4 of membrane insertion and orientation of type 1

•Successive BiP-ADP binding - ratchets nascent protein into ER

Explain step 4 post-translational translocation.

•Elongating polypeptide chain •Passes through the translocon channel into the ER lumen •Signal sequence - cleaved by signal peptidase and rapidly degraded

Explain step 5 of cotranslational translocation

Synthesis continues - elongating chain loops into the cytosol through a small space between the ribosome and translocon

Explain step 5 of membrane insertion and orientation of type 1

•ATP for ADP exchange releases BiP from nascent protein.

Explain step 5 post-translational translocation.

•Growing peptide chain - continues extrusion through the translocon into the ER as the mRNA is translated toward the 3′ end

Explain step 6 of cotranslational translocation

•Synthesis completes at the stop codon - •Ribosomal subunits are released into the cytosol. •Frees the nascent protein to diffuse laterally in the ER membrane

Explain step 6 of membrane insertion and orientation of type 1

•Protein folds into native conformation.

Explain step 6 post-translational translocation.

•Translation completes at mRNA stop codon - ribosome is released

Explain step 7 of cotranslational translocation

•Nascent protein - remainder drawn into the ER lumen and folds into native conformation •Translocon closes.

Explain step 8 of cotranslational translocation

Protein Disulfide Isomerase

Forms and rearranges protein cysteine disulfide bonds

apoptotic

If UPR cannot correct the folding error, the UPR triggers activation of the ____________ pathway

chaperone

Matrix __________________ proteins bind polypeptide chain; helps it refold

dislocation

Misfolded secretory proteins - recognized by specific ER membrane proteins and targeted for transport from the ER lumen into the cytosol for degradation

cytoplasm (free ribosomes) or surface of ER

Most cellular proteins synthesized in 1 of 2 places

Type I proteins

N-terminal signal sequence (cleaved) + single internal stop-transfer anchor (STA) sequence

Topology

N-terminus in ER lumen, single transmembrane domain, C-terminus in cytosol

unfolded protein response

Occurs when quality control system is overwhelmed: misfolded proteins accumulate in ER, for example under certain stresses; presence of unfolded proteins in the rough ER increases transcription of genes that encode ER chaperones and other folding catalysts

protein folding, stability, adhesion, and recognition

Oligosaccharide chains promote what 4 things?

active protein translocators

Proteins bound for the ER, mitochondria/chloroplast and peroxisome are moved across the respective membranes via ....

(-) peptide and (+) peptide, channel helices

Sec61 complex structure

transporter inner membrane (TIM)

Signal sequence inserted through TOM complex, interacts with receptor in the ________________ ___________ __________________ complex. (mitochondrial matrix)

transporter outer membrane (TOM)

Signal sequence recognized by a receptor protein in the ____________ ______________ ______________ complex. (mitochondrial matrix)

unraveled

Such translocation often requires the proteins to be _____________ as they are transported. In the case of the ER, this process occurs as the protein is being translated

true

T/F: C-terminal tail-anchored proteins have no N-terminal signal sequence

true

T/F: Mitochondria and Chloroplast contain their own genome, produce their own ribosomes and synthesize some of their own proteins

nuclear pores

The nuclear envelop possesses large transport complexes that span both membranes

modification

These pores are so large they allow proteins to pass without the need for

orientation

Topogenic sequences determine the ______________ of ER membrane proteins

mutated

Type II and III proteins flip orientation if signal-anchor sequence-flanking positively charged residues are ____________.

•Proteins in cytosol •Peripheral proteins on membranes •Proteins transported into nucleus •Proteins incorporated into peroxisomes, mitochondria and plastids

What proteins are found in the cytoplasm on "free" ribosomes?

cytoplasmic free ribosomes

Where does protein synthesis begin?

-Secreted proteins -Soluble and integral membrane proteins of •ER •Golgi complex •Lysosomes •Endosomes •Vesicles •Plant vacuoles

Which proteins are bound to the surface of the RER?

nuclear proteins

enter and exit through pores in the nuclear envelope

C

except positively charged residues on the signal-anchor sequence _______-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

unfolded state

in step one of protein import into the mitochondrial matrix: proteins are maintained in an _______________ __________ by bound chaperones, such as cytosolic Hsp70

cotranslational translocation

initiated by Signal Recognition Particle (SRP) and SRP receptor GTP-hydrolyzing proteins

channel helices

may separate for lateral passage of a nascent protein hydrophobic transmembrane domain into the ER membrane lipid bilayer

transport vesicles

membrane enclosed sacs that pinch off from, are transported between and fuse with the membranes of endomembrane system organelles; Proteins moving from the ER to other organelles of the endomembrane system are ferried by them

internal signal-anchor sequence

moves laterally through a hydrophobic cleft between translocon subunits to anchor the protein in the ER phospholipid bilayer

Hydrophobic C-terminus

not available for membrane insertion until protein synthesis is complete and the protein has been released from the ribosome

Completion of protein synthesis

releases ribosome subunits into cytosol and peptide C-terminus into the ER lumen

(+) Peptide

ring of isoleucine residues at the constricted waist of the pore - forms a gasket that keeps the channel sealed to small molecules even when translocating a polypeptide (second gating mechanism).

Post-translational translocation

some yeast secretory proteins enter the ER lumen through the Sec61 translocon after translation has been completed (no SRP/receptor involvement)

ribosomes

the rough ER has attached ribosomes actively synthesize proteins

Calnexin and calreticulin

•(lectins) bind to certain oligosaccharide chains (seven N-linked oligosaccharide chains) - helps prevent premature folding into incorrect conformation

Calnexin (CNX) and calreticulin (CRT)

•Bind N-linked oligosaccharides in which a glucose is re-added to form Glc1Man9(GlcNAc)2 (after removal of three glucoses during normal ER processing) •Retain protein in the ER for folding chaperone activity until properly folded

integral membrane proteins

•Classified by orientation in the membrane, locations of N- and C-termini, and the types of targeting signals they contain •Hydrophobic α helices segments - embed in the membrane bilayer •Hydrophilic regions - fold into various conformations outside the membrane

protein targeting

•Delivery of newly synthesized proteins to their proper cellular destinations •Occurs specifically during translation or soon after synthesis

specific transamidase

•GPI-anchored protein formation: •Cleaves the precursor protein within the exoplasmic-facing domain, near the stop-transfer anchor sequence (red) •Covalently links new C-terminus to the terminal amino group of a preformed GPI anchor

protein

•GPI-anchored protein formation: •synthesized and inserted into the ER membrane like a type I transmembrane protein

Glycosylphosphatidylinositol (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.

C-terminus

•If this is a soluble protein once the __________________ has passed through the translocator the signal sequence is cleaved and the protein released

Type II and III proteins

•Lack N-terminal SS •Have a single internal hydrophobic signal-anchor sequence - both ER signal sequence and membrane anchor

Signal-sequence-binding domain

•Large cleft lined with hydrophobic amino acids - binds to signal sequence hydrophobic core

Type IV-B proteins

•No N-terminal signal sequence •N-terminus in the ER lumen •Type III SA sequence followed by alternating type II SA and STA sequences

Type IV-A proteins

•No N-terminal signal sequence •N-terminus in the cytosol •Alternating type II SA sequences and STA sequences

N

•Positively charged amino acids on _________-terminal side of signal-anchor sequence orient nascent polypeptide chain in the translocon with the N-terminal portion in the cytosol.

initiation of protein translation on ribosomes in the cytoplasm with the exception of some proteins produced directly in the mitochondria or chloroplasts.

•Protein sorting is a complex delivery system that utilizes a combination of three processes and always begins with ??

Proteins that can't fold properly

•Retained in the ER for longer times. •Eventually undergo mannose trimming by ER α-mannosidases to form Man5-6(GlcNAc)2 •OS-9 binding to Man5-6(GlcNAc)2 leads to dislocation of the misfolded protein out of the ER, ubiquitinylation, and degradation by cytosolic proteasomes.

translocon

•SRP receptor passes ribosome to a protein translocator (___________) that remains bound to the signal while peptide translation continues into the ER lumen

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 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.

smooth ER

•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 proteins (sarcoplasmic reticulum). •Synthesis of most membrane lipids (ex. Cholesterol).

PDI

•catalyzes formation of disulfide bonds -Properly folded monomer releases folding chaperones

(-) Peptide

•channel closed by a short helical plug - moves out of the channel during protein translocation (one gating mechanism).

Sec61α translocon component

•contacts nascent secretory proteins as they pass through the translocon into the ER lumen.

mitochondrial and chloroplast protein

•imported and localized in multiple organelle compartments by several mechanisms

Type IV proteins

•multiple transmembrane α helices (multipass membrane proteins) •G protein-coupled receptors (shown): seven α helices, N-terminus on the exoplasmic side of the membrane, and C-terminus on the cytosolic side Other type IV proteins - different number of helices and locations of the N- and C-termini

translocon

•preserves integrity of the ER membrane by two gating mechanisms