Week 7 - cell structure

Vesicle Scission

"Dynamin" recruited to the bud neck, cuts bud neck to free vesicle, clathrin cannot cut lipid bilayer -costs energy, GTPase -absence of hydrolizable GTP

Transport and Targeting

"Rab proteins" act as address labels for vessicles -another class of GTPase proteins -GTP binding causes amphipathic helix to become accessible (like SAR1) -more than 60 known Rabs, different rabs target different organelles -rabs bind: --> motor proteins, mediate movement --> targeting, act to localize vesicle

Example of Constitutive Internalization

"housekeeping": CHOLESTEROL -> hydrophobic -in order for it to travel in blood to get to cells it must be modified "LDL" low density lipoprotein, transports cholesterol in blood -cholesterol esterified to long chain fatty aids -cholesterol core surrounded by: a. single layer phospholipid b. copy of cipolipoprotein B100 LDL receptor taken up whether cargo bound or not ALWAYS cycling. (constitutive) *mutant form of LDL puts people @ risk, 5 forms of mutaions

Protein Modifications in Golgi Complex

"phosphorylation" add phosphate, at cis "glycosylation" N and O linked "sulfation" add sulfate, trans, at trans

Proteins in ERC

-> proteins can be recycled from endosomal compartments -> early endosome are not actively digesting compartments (pH is not acidic enough) -> proteins can be shuffled from one cell surface to another through transcytosis

Major Proteins in Synaptic Vesicles

-Vsnare (fusion) -synaptotagmin (calcium sensor) -vATPase (pH) -transporter (neurotransmitter)

Steps in Vesicle Formation (Cargo recruitment, Budding)

-adaptor proteins bind cargo receptors/integral membrane proteins -adaptor proteins recruit coat proteins -membrane forms the vesicle 1.cargo receptors bind specific proteins to be trafficked 2. adaptor proteins bind to cargo receptor proteins 3. adaptor protein binding concentrates cargo receptors, thus recruits cargo to new bud 4. adaptor proteins recruit coat protein to induce membrane curvature

Retention System for Maintaining Composition

-based on physical properties of proteins prevent them from entering vesicles -some proteins escape

Cisternal Maturation Model for IntraGolgi Transport

-cargo in stack never moves -maturation of stack -need vesicles for enzyme retrieval -from cis to trans

Clathrin

-composed of heavy and light chains -stable basic unit is triskelion (3 heavy, 3 light) -triskelions function as basic unit for higher order clathrin assembly (cage or lattice) -coat property -->cage assembly contributes to vesicle formation, NO pinching

"Fusion Pore"

-connection between outside and lumen of vesicle

Constitutive Exocytosis Pathway

-default pathway for proteins that have no sorting signal other than the ER signal sequence and exit signal -brings proteins to be secreted or to plasma membrane

Secretory/Large Dense Core Vesicles

-different tissue/cell types (endocrine vs. exocrine) -different sizes/content (insulin vs. chromaffin granules)

Visualizing Exocytosis

-electron microscopy, but it is static -light microscopy, live imagine of fluorescently labeled proteins, total internal reflection is fluorescent ---> found different types of regulated exocytosis: 1. full fusion 2. kiss and run 3. kiss and stay

Caveolae Mediated Endocytosis

-elongated flask shaped structure in plasma membrane (50-80 nm) composed of distinct proteins/lipid: -caveolins/cavins (coat), GPI-anchored proteins (cargo) -lipid rafts (cholesterol, sphingolipids) **unusual lipid composition of lipid rafts believed to recruit cargo not cargo receptors -scission through dynamin -abundant in endothelial cells, apipocytes, muscle cells -signal transduction, lipid regulation, mechanosensing

"Macrophinocytosis"

-engulfment of large gulps of membranes -irregular shape -actin-dependent process, from surface membrane ruffles -triggered by growth factors -exploited by various pathogens (HIV, vaccina, HSV, aeno3)

Vesicular Transport

-folded proteins ready to exit, "membrane trafficking," must have a way to select cargo

Clathrin Mediated Endocytosis

-form rapidly, ~1 min to make located pit -thousands of clathrin coated vesicles leaving cell surface every minute -size of clathrin coated vesicle 30-200 nm -scission through dynamin

Organelles Undergoing Exocytosis

-late endosome/lysosome -constitutive secretory vesicles -transported secretory vesicles: 1. secretory granules 2. synaptic vesicles

Types and Characteristics of Endosome

-membrane bound organelle Types: early endosome, recycling endosome, multi-vesicular bodies/late endosome, endolysosome---lysoosome Characteristics: particular protein, lipid content, pH, time it takes for endocytosed material to reach it, different morphology --> can represent same organelle at different stages of its life

Vesicle Fusion

-membrane bound organelle fuses -mediated on vesicle and organelle by SNARE proteins "v-snare" (vesicle) and "t-snare" (target) 1 "v-snare" and 3 "t-snare" zip together causing them to fuse (4-helix bundle)

Coat Formation and GTPase

-must get rid of the coat for fusion to take place -GTPase can regulate coat formation: ARF1 (GTPase) regulate Cathrin/COPI assembly SAR1 (GTPase) regulates COPII assembly -Cargo receptor proteins function as a GEF for SAR1/ARF1 -SAR1-GTP extends amphipathic helix to embed protein in lipid bilayer -assist recruitment of adaptor + coat proteins

Endocytic Pathways Permit....

-nutrient uptake -maintenance of cell membrane -regulation of cell signaling (receptors @ plasma membrane) *hijacked by pathogens

Location of Retrieval System and KDEL Receptor

-opperates in VTC and Golgi pH is neutral in ER, no binding for KDEL, only releasing pH acidic in Golgi w/proton pumps, binding for KDEL

Exocytosis

-process by which cells release material outside such as lipids, soluble proteins, membrane proteins,

Endocytosis

-process by which materials get INTO the cell

Retrieval System for Maintaining Composition

-proteins that have sorting signals that will signal whether they should be kept and/or returned to ER "ER resident membrane proteins": KKXX at C-terminus, functions to directly bind to/recruit COPI coat proteins "ER resident soluble proteins": KDEL at C-terminus, KDEL sequence cannot bind COPI coat proteins, so there must be KDEL receptor (another step), receptor binds to COPI coat proteins

Regulated Exocytosis

-proteins travel to plasma membrane after being made -primed for fusion (V-snare/T-snare may be associated but don't make 4-helix bundle) -fuse and release on signal (entry of Ca2+) -complexin prevents fusion, but the synaptotagmin senses and binds to the calcium, releasing complexin clamp -can expand cell surface area examples: neurotransmitter, neuropeptides, peptide hormones, growth hormones

Lysosome

-site of intracellular digestion -heterogenous (different sizes/shapes) -found in mammalian cells -receive inputs from both biosynthetic endocytic pathway -mechanism of macromolecules degradation lysosome (degrades many macromolecules)/proteasome (only degrades proteins); garbage disposal

GLUT4 in the Endoplasmic Recycling Compartment

-stored in specialized recycling endosomes in adipocytes and muscle cells -can be mobilized when needed (insulin signaling) -recycling transcytosis (endo and exo)

Late Endosome/Multi-vesicular Bodies

-transition/maturation from early endosome to a lysosome -homotypic fusion of early endosome, increases size -budding of intralumenal vesicles -change in protein composition (RAB5- RAB7) -Decrase pH (4.5-5.5) -lack of tubules -same process as HIV, influenza,ebola to bud from cells -depends on escort proteins "monoubiquition" labels transmembrane proteins for degradation

Vesicular Transport Model for IntraGolgi Transport

-transport vesicles through golgi from distinct cisternal compartments

N-Linked Glycosylation at Golgi

-very complex -different enzymes, different stacks -enzymes associated with stacks

ER -> Golgi Transport

-vesicular tubular clusters are an important intermediate in ER to Golgi trafficking MUST be sorting signals on all proteins that will be recognized for transport out of ER -as vesicles approach golgi they fuse together "homotypic fusion" (ERGIC) -lumenal cargo protein, transmembrane cargo protein, cargo receptor -cargo receptors activate SAR1 through GEF activity -adaptors and COPII coat recruited

Steps in Vesicular Transport

1. Make vesicle, recruiting cargo. CAN be soluble, CAN be integral membrane cargo (protein) 2. Vesicle scission 3. Transport and targeting 4. Tethering 5. Fusion

Main Endocytic Pathways

1. Phagocytosis, regulated process (~cell eating, entire bacteria) 2. Pinocytosis (~cell drinking) a. Bulk Phase, constitutive process, happens all the time. "macrophinocytosis" b. Receptor-Mediated, constitutive and regulated process

Examples of Ligand Induced Internalization

1. Receptor tyrosine kinases: EGF epidermal growth factor 2. GPCR: g-protein coupled receptor -endocytosis regulates signaling by controlling the number of receptors at the plasma membrane -MOSTLY clathrin dependent

Why glycosylate?

1. can be used as target motiff, sorting signals 2. special structural to proteins -sugars, most rigid macromolecules -adding sugars fends off other proteins -protects proteins from protease -keeps bacteria from cell surface

3 Vesicle Types

1. clathrin - coated vesicles: trafficking from golgi, plasma membrane 2. COPI - coated vesicles: trafficking at golgi complex 3. COPII - coated vesicles: trafficking from ER, first vesicle proteins see

3 Types of Membrane Ruffles

1. lamellipodia-like "flat" 2. circular ruffles, "cup-shaped" 3. blebs

GTPase and Disassembly of Coat

BUT GTPase can function as timers, regulation duration of coated vesicle.... -SAR1/ARF1 proteins will hydrolyze GTP, become inactive, pull in helix, coat/adaptor proteins fall off vesicle

Constitutive Secretory Pathway vs. Regulated Secretory Pathway

Constitutive: unregulated, example is GFP marked protein Regulated: requires a signal, leads to intracellular Ca2+ concentration

Receptor Mediated Endocytosis

a way to internalize specific molecules/selective uptake: -receptor binds to specific cargo -receptor binds to adaptor -adaptor interacts with coat protein -constitutive v ligand induced internalization... (housekeeping vs. signaling) 1. Clathrin-mediated 2. Caveolae-mediated 3. Caveolin-independent/Clathrin-independent

Trans Golgi Network

acts as a "sorting station": -late endosome/lysosome -plasma membrane/secretion 1. constitutive 2. regulated -vesicle/tubular carriers -protein routing depends on lumenal interactions and cytosolic trafficking sequence

Caveolin-independent and Clathrin-independent BUT STILL mediated processes

example: F-actin dependent -polymerization of F-actin drives elongation and scission of invaginated membrane

Secretory Granules Formation

granulogenic proteins - tendency to aggregate TGN (trans golgi network) environment, aggregate at low pH, redox, coat is important for maturation

Synaptic Vesicles

mediate release of neurotransmitters at synapse -form locally, form endocytic vesicles -very similar mechanisms to secretory granules in exocytosis

Early endosome acts as....

sorting solution for internalized proteins/lipids: -plasma membrane is direct (fast) or through recycling endosome (slow) -lysosome (degradation) -TGN (retrograde transport)

Golgi Complex

stacked structure, flattened, disklike cisternae (cis/trans face) "cis": where proteins enter "trans": where proteins exit functions: protein modification post translation protein sorting

Tethering

tethering proteins bound to target compartment: -recognize incoming vesicle -assemble into long, multiprotein complexes -bind to rab and localize vesicle

"early endosome" characteristics

usually near periphery, first organelle visited by endocytic vesicles, focal point determined by different endocytic pathways -RAB5 -PI3P -TUBULES -pH 6.0-6.5