INTD 5000 Lectures C 7 C 8

INTD 5000, Lectures C 7 & C 8 Professor Eileen M. Lafer Tel#: 7 -3764 Email: Lafer@biochem. uthscsa. edu Office: Room 415 B THE SECRETORY AND ENDOCYTIC PATHWAYS Reading: Chapters 12 and 13 from Alberts et al. , Molecular Biology of the Cell

CELL COMPARTMENTS (ANIMATION 12. 1 ALBERTS)

THREE TYPES OF PROTEIN MOVEMENT BETWEEN COMPARTMENTS Gated transport: nuclear pore complex, Transmembrane transport: protein translocators (proteins usually unfold), cytosol<-> nucleus cytosol-> ER, cytosol-> mitochondria Vesicular transport: membrane-enclosed transport, ER <-> golgi post-golgi traffic Alberts

THE BIOSYNTHETIC-SECRETORY AND ENDOCYTIC PATHWAYS Alberts

ENDOPLASMIC RETICULUM (ER): An extensive tubovesicular network where proteins and lipids are made. Rough ER: studded with ribosomes, site of protein biosynthesis Smooth ER: site of lipid biosynthesis Alberts

ROUGH ER Alberts

SMOOTH ER Alberts

Alberts

PROTEIN SYNTHESIS AND TRANSLOCATION IN THE ROUGH ER Animation 12. 6, Alberts

PROTEIN GLYCOSYLATION IN THE ROUGH ER: During translation, a signal sequence on membrane and secretory proteins directs the nascent protein into the ER lumen. After the protein has entered the ER, the glycosylation process begins.

A PRE-FORMED PRECURSOR OLIGOSACCHARIDE IS TRANSFERRED EN BLOC TO PROTEINS IN THE ER Alberts

PROTEIN GLYCOSYLATION IN THE ROUGH ER Alberts

SOME PERIPHERAL MEMBRANE PROTEINS AQUIRE A COVALENTLY ATTACHED GLYCOPHOSPHATIDYLINOSITOL (GPI) ANCHOR IN THE ER Alberts

THE BIO-SYNTHETIC SECRETORY AND ENDOCYTIC PATHWAYS Retrieval pathways are in blue. Alberts

MOVIE: MOVEMENT OF A FLUORESCENTLY TAGGED MEMBRANE PROTEIN THROUGH THE SECRETORY PATHWAY Alberts Video 13. 2

EXOCYTOSIS AND ENDOCTYOSIS Alberts

VESICULAR TRANSPORT Transport vesicles bud from one compartment and fuse with another, carrying material from the lumen of the donor compartment, and depositing it in the lumen of the target compartment. Alberts

VESICULAR TRANSPORT IS MEDIATED BY COATED VESICLES Alberts et al. , Molecular Biology of the Cell-3 rd edition

THERE ARE VARIOUS TYPES OF COATED VESICLES Alberts

DIFFERENT COATS ARE USED IN DIFFERENT TRAFFICKING PATHWAYS Alberts

CLATHRIN-COATED VESICLES ARE THE MOST WELL CHARACTERIZED TRANSPORT VESICLES John Heuser

THE STRUCTURE OF A CLATHRIN TRISKELION Ungewickell and Branton

THE STRUCTURE OF A CLATHRIN COAT Kirchhausen, Harrison, Walz, Fotin

The Assembly and Disassembly of a Clathrin Coat

THE CLATHRIN ASSEMBLY PROTEINS Monomeric AP Family AP 180 Synaptic Plasma Membrane CALM Ubiquitous Tetrameric AP Family (also called adaptins) AP-1 AP-2 AP-3 AP-4 AP 180 N AP-2 C b Clathrin assembly PIP binding TGN Plasma Membrane Endosome/Lysosome TGN PIP binding m s

Adaptins (AP-1, AP-2, AP-3, AP-4) Select Cargo For Inclusion Into Coated Vesicles, and Promote Clathrin Polymerization YXXF-m 2 LL-b 1 NPXY-clathrin TD Alberts et al. , Molecular Biology of the Cell

AP 180 Promotes the Formation of Homogeneously Sized Vesicles Clathrin Cages Assembled in vitro Without AP 180 Clathrin Cages Assembled In vitro With AP 180 Ye & Lafer, 1995 Synaptic Vesicles in Drosophila Lacking Fly AP 180 Gene lap Synaptic Vesicles in Wild Type Drosophila Containing Fly AP 180 Gene lap Zhang, Koh, Beckstead, Budnick, Ganetzky, & Bellen, 1998

AP 180 recruits clathrin to the membrane via interactions with the phosphoinositide PIP 2, and stimulates coat formation. Diameter of lattice: 66 nm Matthew Higgins and Harvey Mc. Mahon

THE GTPase DYNAMIN PROMOTES MEMBRANE SCISSION Alberts

How do clathrin coated vesicles uncoat? Hsc 70 auxilin clathrin APs Ca++

How do clathrin coated vesicles uncoat? Disrupt Interaction Between Hsc 70 and Auxilin Hsc 70 Auxilin-DHPD clathrin APs Ca++

Auxilin DHPD Increases the Number of Coated Vesicles by 6 Fold: Auxilin DHPD * * J. R. Morgan, K. Prasad, S. Jin, G. J. Augustine, and E. M. Lafer. Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals: Roles for Hsc 70 and Auxilin. Neuron 32: 289 -300 (2001).

Auxilin Recruits and Activates the Uncoating ATPase Hsc 70 to Clathrin Coated Vesicles Hsc 70 auxilin clathrin APs Ca++

The Clathrin Coated Vesicle Cycle

CLATHRIN – THE MOVIE ANIMATION 13. 1 ALBERTS

ASSEMBLY AND BUDDING OF COPII and COPI COATED VESICLES FROM: Lee et al. , Annu. Rev. Cell Dev. Biol. 20: 87 -123, 2004.

STRUCTURE OF THE SEC 13/31 COPII COAT FROM: Stegg et al. , Nature 439: 234 -239, 2006.

MOVIE: STRUCTURE OF THE SEC 13/31 COPII COAT FROM: Stegg et al. , Nature 439: 234 -239, 2006.

ORIENTATION OF THE SEC 13/32 HETEROTETRAMER IN THE SELF-ASSEMBLED OCTAHEDRAL CAGE FROM: Stegg et al. , Nature 439: 234 -239, 2006.

SNARE PROTEINS CONTRIBUTE TO THE SELECTIVITY OF VESICLE-TARGET DOCKING AS WELL AS TO THE FUSION PROCESS

SNARE PROTEINS: The group of proteins commonly referred to as SNARES for Soluble NSF Attachment REceptor. S were originally discovered as the synaptic proteins: VAMP/synaptobrevin syntaxin SNAP-25 They were later re-discovered as the receptors for the soluble Golgi trafficking protein SNAP – Soluble NSF Attachment Protein. NSF (NEM-Sensitive Fusion factor) The SNARE proteins were also identified as substrates for the clostridial neurotoxins, potent agents which inhibit neurotransmitter release.

The SNARE Complex: The synaptic vesicle membrane protein synaptobrevin (v-snare), forms a tight ternary complex with the presynaptic plasma membrane proteins syntaxin (tsnare), and SNAP-25 (t-snare). The stoichiometry of the proteins in the complex is 1: 1: 1 and it is resistant to SDS. This complex can be actively disassembled by the ATPase NSF, together with -SNAP.

FROM: Sutton et al. , Nature 395: 347 -353, 1998. Topology and organization of the synaptic fusion complex. a, Backbone ribbon drawing of the synaptic fusion complex: blue, synaptobrevin-II; red, syntaxin-1 A; green, SNAP-25 B (Sn 1 and Sn 2).

Hypothetical model of the synaptic fusion complex as it joins two membranes, and location of neurotoxin-mediated cleavage sites. We extended the synaptic fusion complex crystal structure to include the transmembrane domains (yellow) of syntaxin-1 A (red) and synaptobrevin-II (blue), and the loop connecting the Sn 1 and Sn 2 fragments (green). The transmembrane domains and the linker to the Sx fragment are represented as a-helices. Hypothetical bends of the syntaxin and synaptobrevin a-helices were modelled close to the lipid bilayers. The loop between the Sn 1 and Sn 2 fragments was modelled as an unstructured polypeptide chain. The conformation of this loop is speculative. The loop between the Sn 1 and Sn 2 domains is shown in orange.

A MODEL FOR HOW SNARE PROTEINS MAY CATLYZE MEMBRANE FUSION Alberts

DISSOCIATION OF SNARE PAIRS BY NSF FOLLOWING A CYCLE OF MEMBRANE FUSION Alberts

RAB PROTEINS ARE SMALL GTPases THAT GUIDE VESICLE TARGETING Alberts

TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS Alberts

PROTEINS LEAVE THE ER IN COPII-COATED TRANSPORT VESICLES

ER RESIDENT PROTEINS CONTAIN A KDEL SEQUENCE THAT INTERACTS WITH THE COPI COAT AND PERMITS THEIR RETRIVAL FROM THE GOLGI BACK TO THE ER

A MODEL FOR THE RETRIEVAL OF SOLUBLE ER RESIDENT PROTEINS

THE GOLGI APPARATUS CONSISTS OF AN ORDERED SERIES OF COMPARTMENTS Alberts

THE TRANSITIONAL ZONE BETWEEN THE ER AND GOLGI Alberts

THE GOLGI APPARATUS Alberts

OLIGOSACCHARIDE CHAINS ARE PROCESSED IN THE GOLGI APPARATUS

TRANSPORT INTO THE CELL FROM THE PLASMA MEMBRANE: ENDOCYTOSIS

PINOCYTIC VESICLES FORM FROM COATED PITS IN THE PLASMA MEMBRANE 1. The major type of endocytic vesicles are the clathrin coated vesicles (CCVs). The CCVs shown below are from a hen oocyte taking up lipoprotein particles to form yolk: Alberts

2. Caveolae are a less well understood type of endocytic coated vesicle that are involved in the endocytosis of lipid rafts from the plamsa membrane. Their major structural proteins are caveolins which are integral membrane proteins. Alberts

ENDOCYTOSIS IS IMPORTANT FOR CELLS TO: 1. Import selected extracelluar molecules (i. e. receptor-mediated endocytosis). 2. Regulate levels of membrane proteins on the cell surface (i. e. receptor down-regulation). 3. Synaptic vesicle recycling and biogenesis.

EXAMPLE: RECEPTOR MEDIATED ENDOCYTOSIS Cholesterol molecules are packaged in LDL particles. Alberts

RECEPTOR-MEDIATED ENDOCYTOSIS: THE MOVIE Animation 13. 3 from Alberts

EXOCYTOSIS Alberts

SECRETORY VESICLES BUD FROM THE TRANS-GOLGI Alberts

EXOCYTOSIS OF SECRETORY VESICLES The release of insulin from a secretory vesicle of a pancreatic beta cell. Alberts

SYNAPTIC TRANSMISSION IS AN EXAMPLE OF REGULATED EXOCYTOSIS SYNAPTIC TRANSMISSION IS ALSO AN EXAMPLE OF A COUPLED EXOENDOCYTIC CYCLE Augustine