INTD5000, Lectures C7 & C8Professor Eileen M. Lafer

Tel#: 7-3764

Email: Lafer@biochem.uthscsa.edu

Office: Room 415B

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, cytosol<-> nucleus

Transmembrane transport: protein translocators (proteins usually unfold), cytosol-> ER,

cytosol-> mitochondria

Vesicular transport: membrane-enclosed transport, ER <-> golgi

post-golgi traffic Alberts

THE BIOSYNTHETIC-SECRETORY ANDENDOCYTIC 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

Alberts

Retrieval pathways are in blue.

MOVIE: MOVEMENT OF A FLUORESCENTLY TAGGEDMEMBRANE PROTEIN THROUGH THE SECRETORY PATHWAY

Alberts Video 13.2

EXOCYTOSIS AND ENDOCTYOSIS

Alberts

VESICULAR TRANSPORT

Alberts

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.

VESICULAR TRANSPORT IS MEDIATED BY COATED VESICLES

Alberts et al., Molecular Biology of the Cell-3rd 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

Ungewickell and Branton

THE STRUCTURE OF A CLATHRIN TRISKELION

THE STRUCTURE OF A CLATHRIN COAT

Kirchhausen, Harrison, Walz, Fotin

The Assembly and Disassembly of a Clathrin Coat

Monomeric AP Family Tetrameric AP Family (also called adaptins)

Clathrin assembly

N C

AP180 AP-2

Clathrin assembly

AP180 Synaptic Plasma Membrane AP-1 TGNCALM Ubiquitous AP-2 Plasma Membrane

AP-3 Endosome/LysosomeAP-4 TGN

PIP bindingPIP binding

THE CLATHRIN ASSEMBLY PROTEINS

Adaptins (AP-1, AP-2, AP-3, AP-4) Select Cargo For Inclusion Into Coated Vesicles, and Promote Clathrin

Polymerization

Alberts et al., Molecular Biology of the Cell

YXX

LL

NPXY-clathrin TD

AP180 Promotes the Formation of Homogeneously Sized Vesicles

Clathrin Cages Assembled in vitro Without AP180

Clathrin Cages Assembled In vitro With AP180

Synaptic Vesicles in DrosophilaLacking Fly AP180 Gene lap

Synaptic Vesicles in Wild Type DrosophilaContaining Fly AP180 Gene lap

Ye & Lafer, 1995 Zhang, Koh, Beckstead, Budnick, Ganetzky, & Bellen, 1998

AP180 recruits clathrin to the membrane via interactions with the phosphoinositide PIP2, and stimulates coat formation.

Matthew Higgins and Harvey McMahon

Diameter of lattice: 66nm

THE GTPase DYNAMIN PROMOTES MEMBRANE SCISSION

Alberts

How do clathrin coated vesicles uncoat?

clathrinAPs

Ca++

auxilinHsc70

How do clathrin coated vesicles uncoat?

clathrinAPs

Ca++

Auxilin-HPDHsc70

Disrupt Interaction Between Hsc70 and Auxilin

Auxilin HPD Increases the Numberof Coated Vesicles by 6 Fold:

0

1

2

HPDAuxilin Control

#CV

s / A

Z

Auxilin

** **

Auxilin HPD

J.R. Morgan, K. Prasad, S. Jin, G.J. Augustine, and E.M. Lafer. Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals: Roles for Hsc70 and Auxilin.

Neuron 32: 289-300 (2001).

Auxilin Recruits and Activates the Uncoating ATPase Hsc70 to Clathrin Coated Vesicles

clathrinAPs

Ca++

auxilinHsc70

The Clathrin Coated Vesicle Cycle

CLATHRIN – THE MOVIEANIMATION 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 SEC13/31 COPII COAT

FROM: Stegg et al., Nature 439: 234-239, 2006.

MOVIE: STRUCTURE OF THE SEC13/31 COPII COAT

FROM: Stegg et al., Nature 439: 234-239, 2006.

ORIENTATION OF THE SEC13/32 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 forSoluble NSF Attachment REceptorS

were originally discovered as the synaptic proteins:VAMP/synaptobrevin

syntaxinSNAP-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 (t-snare), 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-1A; green, SNAP-25B (Sn1 and Sn2).

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-1A (red) and synaptobrevin-II (blue), and the loop connecting the Sn1 and Sn2 fragments (green). The transmembrane domains and the linker to the Sx fragment are represented as -helices. Hypothetical bends of the syntaxin and synaptobrevin -helices were modelled close to the lipid bilayers. The loop between the Sn1 and Sn2 fragments was modelled as an unstructured polypeptide chain. The conformation of this loop is speculative. The loop between the Sn1 and Sn2 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

Alberts

TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS

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 RETRIVALFROM 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

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 MOVIEAnimation 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 REGULATEDEXOCYTOSIS

 SYNAPTIC

TRANSMISSION IS ALSO AN

EXAMPLE OF A COUPLED

EXO-ENDOCYTIC

CYCLEAugustine