Molecular cell biology

MOLECULAR CELL BIOLOGY

• MOLECULAR CELL BIOLOGY

• SIXTH EDITION

13.1 Techniques for studying the secretory pathway Genetic and Biochemical Approaches

1. Transport of a protein through the secretory pathway can be assayed in the living cells

a. G. Palade pancreatic acinar cell: pause-chase and autoradiography 5 min “hot” → “cold” for 1, 5, 10, 30, 60, 120 min b. visualizing by fluorescence microscopy

a. G. Palade pancreatic acinar cell: pause-chase and autoradiography 5 min “hot” → “cold” for 1, 5, 10, 30, 60, 120 min b. visualizing by fluorescence microscopy

a. Construct genes containing GFP + VSVG (vesicular stomatitis virus glycoprotein, mutant version) and transfect into cultured mammalian cells b. Synthesize VSVG on ER c. At 40 Co, VSVG is misfolded and retained in ER, at 32 Co, VSVG is folded correctly, and transferred to Golgi and plasma membrane.

An another way to follow the transport of secretory proteins: modifications of CHO side chains at different stages of secretory pathway

• 2. Detection of compartment- specific

• oligosaccharide modifications

3. Yeast mutants define major stages and many components in vesicular transport Many secretory vesicles stay in the space between membrane and cell wall (invertase, sucrose → glucose and fructose). Use temp sensitive mutant (sec) or double mutant to detect the accumulation protein in ER or Golgi

4. Cell-free transport assays allow dissection of individual steps in vesicular transport proteins (enzymes) transport from one Golgi cisterna to another needs cytosolic extract, ATP, GTP, physiological temperature

14.2 Molecular mechanisms of vesicular traffic 1999 Nobel Prize Award: Gunter Blobel donor organelle → target organelle vesicle budding and fusion

Binding : 1. recruitment of a small GTP-binding protein to a patch of donor membrane coat protein 2. membrane cargo protein involving membrane cargo-receptor protein 3. V-SNARE 4. soluble proteins

Assembly of a protein coat drives vesicle formation and selection of cargo molecules

COP II: RER → Golgi COP I: (retrograde) Golgi-Golgi cisternae, cis- Golgi → RER Clathrin: Trans Golgi →late endosome PM →late endosome Trans-Golgi → plasma membrane ??

2. A conserved set of GTPase switch proteins control assembly of different vesicle coats Small GTP-binding protein (GTPase) closed related to assembly COP I, Clathrin: ARF COP II : Sar1

Model for the role of Sar1 in the assembly and disassembly of COP II coats

3. Targeting sequences on cargo proteins make specific molecular contacts with coat proteins membrane sorting signals on membrane cargo proteins and different sorting signals for soluble cargo proteins called luminal sorting signals

SNARE protein: (Soluble NSF Attachment Receptor) T-SNARE: Target SNARE,(syntaxin, SNAP-25) V-SNARE: Vesicle SNARE (VAMP) 60 members in yeast and mammalian cells R-SNARE: an arginine in the formation of SNARE core (synaptobrevin) Q-SNARE: a glutamine of SNARE core (syntaxin, SNAP-25)

• In lysosome study, fusion yeast and

• eukaryotes have 20+ V-SNAREs and

• T-SNAREs

• 2. Mutant study found that a particular vesicle has its own SNAREs (special target sequence)

14.3 Early stages of secretory pathway closer look at vesicular traffic through ER and Golgi (antero-grade and retrograde)

1. COPII vesicles mediate transport from the ER to the Golgi cell-free extracts of yeast RER incubated with cytosol, ATP, nonhydrolyzed analog of GTP → vesicles formed in ER→ proteins for coat polymerization From mutation study, there are several proteins involved in the polymerization

Contain a di-acidic sorting signal that binds to the Sec 24 subunit of the COP II coat for transport out of the ER mutation on position on 508 can not bind to Sec 24, and prevents normal transport of CFTR to the plasma membrane and retained in ER

• Cystic fibrosis : mutations of CFTR (ATP binding

• cassette protein on the epithelium of lung, pancreas, sweat

• glands etc.)

2. COPI vesicles mediate retrograde transport within the Golgi and from the Golgi to the ER ER resident proteins (Bip, isomerase) have KDEL (lys-asp-x-x) at C-terminal, if escape from ER to Golgi, COPI can bring back to ER

• First discovered when isolated Golgi incubated with solution containing ATP, cytosol, and a nonhydrolyzing analog of GTP.

• Isolated vesicles and found they contain 7 polypeptide subunits (coatmers)

• Temperature sensitive mutant to study the functions of proteins

3. Anterograde transport through Golgi occurs by cisternae progression vesicles transport model → cisternal progression model enzymes from trans-Golgi to medial-Golgi, and from medial- to cis-Golgi EM studies

Vesicle-mediated trafficking from Trans-Golgi network

• 14.4 Later stages of the secretory

• pathway

Vesicles coated with clathrin and/or adaptor proteins mediate several transport steps clathrin: fibrous clathrins inner layer: adaptor protein complexes (AP) Cyro-electron mocrographs of 1000 assembly particles ↓ digital imaging

• Cargo protein with tyr-x-x (hydrophobic aa) recruite

• into clathrin/Ap1 and budding from trans Golgi

• to plasma membrane

2. Dynamin is required for pinching off of clathrin vesicles using nonhydrolyzable derivative of GTP or dynamin mutants

3. Mannose-6-phosphate residues target soluble proteins to lysosomes M-6-P is synthesized in the cis-Golgi (N-linked)

4. Study of lysosomal storage diseases revealed key components of the lysosomal sorting pathway I-disease : lack of N-acetylglucosamine phosphotransferase (can’t form M-6-P) in lysosomes → glycolipids become large inclusion bodies and accumulate in the cell.

• 5. Protein aggregation in thr trans-Golgi may

• function in sorting proteins to regulated

• secretory vesicles

• ACTH, insulin, trypsinogen aggregates

• Chromogranin A, Chromogranin B,

• Secretogranin II

• 6. Some proteins undergo proteolytic

• processing after leaving the trans-Golgi

• proproteins: soluble lysosomal enzymes, membrane proteins (influenza hemagglutinin),

• secretion protein (albunim, insulin glucagon, yeast mating factor)

7. Several pathways sort membrane proteins to the apical or basolateral region of polarized cells

Polarized cells contain apical, basolateral, and tight junctions small intestine: absorption stomach: acidification fractionation and microscopic observations : all these proteins from Trans-Golgi→ various kinds of vesicles, different Rab, v-SNARE → targets MDCK (Madin-Darby Canine Kidney)

14.5 Receptor-mediated endocytosis and the sorting of internalized proteins 1. phagocytosis: macrophage 2. invaginated vesicles : a. pinocytosis b. receptor-mediated endocytosis (LDL, insulin, transferrin involving clathrin/AP2)

1. Receptors for LDL and other ligands contain sorting signals that target them for endocytosis

LDL receptor: 839 residues of glycoprotein , signal transmembrane segment, short C-terminal (cytosol, sorting sequence) longer N-terminal (extracellular) containing 7 cysteine-rich which bind to Apo B-100

Familial hypercholesterolemia: LDL receptor (LDLR) → mutation (can’t form receptor) → LDLs internalization decreases and LDL concentration in plasma increases Homozygous: 6X plasma LDL as high as normal, die of heart attack before late 20s also mutation on C-terminal NPXY (Asn-Pro-X-Tyr) sorting sequence → can not bind to AP2 complex → clathrin coat can not form (no pits form) other kinds of receptor mutations can not bind to LDL

2. The acid pH of late endosomes causes most receptor-ligand complexes to dissociate

Model for pH-dependent binding of LDL particles by the LDL receptor

3. The endocytic pathway delivers iron to cells without dissociation of the receptor-transferrin complex in endosomes transport iron from the liver and from the intestine to all the tissues of the body

• 14.6 Directing membrane proteins

• and cytosolic materials to the

• lysosome

Cargo proteins destined to enter the multivesicular endosome usually receive their ubiquitin tag at the PM, the TGN, or the endosomal membrane (cytosolic or misfolded ER proteins – ubiquitine to proteosomes) In the membrane of the endosome, a ubiquitin-tagged peripheral membrane protein, Hrs, facilitates loading ubiquitin tag cargo protein into vesicle buds directed into the interior of the endosome.

• Inward budding of the endosomal membrane

1. Retroviruses bud from the plasma membrane by a process similar to formation of multivesicular endosomes

2. The autophagic pathway delivers cytosolic proteins or entire organelles to lysosomes a. starvation b. autophagy (eating oneself) double membrane structure (from ER?) envelops an entire organelle to form autophagosome c. The autophagosome is organelle specific (signal binding sites?) d. Atg8 protein e. targeting and fusion proteins (?)