Lecture 3Vesicular Trafficking-Cops and Clathrins

-Arfs, Rabs, Sars-Snares

Vesicular Trafficking allows Proteins and Vesicles to Reach their Destinations

The orientation of transmembrane proteins

Proteins IN the ER face the OUTSIDE of the cell

Exocytosis

Vescles are transported along the Actin Cytoskeleton

COPII = ER-> GolgiCOPI = Golgi to Golgi

Vesicular Trafficking allows Proteins and Vesicles to Reach their Destinations

The orientation of transmembrane proteins

Proteins IN the ER face the OUTSIDE of the cell

COPII = ER-> GolgiCOPI = Golgi to Golgi

Vesicle budding and vesicle fusion are two different processesBoth mechanically and mechanistically

Triskeleion = 3 large and 3 smallpolypeptides

= 1 clathrin

Receptor-mediated Endocytosis requires clathrin

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Adaptin binds clathrin and receptors, acting as a bridge (4 types)Hsp70 chaperone and auxillin uncoat the vesicle.What prevents uncoating at membranes?Vesicles form at many membranes (Golgi, PM)At these membranes COPI and COPII function instead of clathrinVesicles can be tubular

Dynamin forms a ring around

the bud (GTPase)

GAP = GTPase Activating ProteinGEF = Guanine Nucleotide Exchange Factor

GTPases function in vesicle formation

GTPases are required for vesicle formation (start) andVesicle fusion (end)

ARF is the GTPase for COPI and Sar is the GTPase for COPIIGEFs determine when the vesicle is ready to budGEF activation triggers GTPase activation and hydrophobic tail exposureGAPs triggers GTPase inactivation. It falls off the membrane and triggers coat disassembly

Snares guide vesicular transportVesicle-surface markers that direct vesicles to the correct place

V=vesicle and t=target SNARES

Snares are integral-membrane proteins that pull membranes together.

Neuronal snares are the targets of neural toxin proteases (botulism)

The coiled coil

The coiled-coil is a tightly intertwined set of 4 -helix domainsThree are contributed by t-snares, and 1 by the v-snareAt least 1 of the t-snares is an integral-membrane proteinSnares (20) and Rabs

Snares also promote Membrane fusion

30 members -each bind a particular vesicle -On the cytoplasmic faceRabs interact with SnaresVariation in Rab C-terminal tailsVariation in effectorsRabs are different from coatAssembly GTPases (ARFs)

Rab GTPases ensure the specificity of vesicular docking

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

SMAP2, a Novel ARF GTPase-activating Protein, Interacts with Clathrin and Clathrin Assembly Protein and Functions on the AP-1–positive Early Endosome/Trans-Golgi Network Waka Natsume et al.

How are Snares separated?NSF is an ATPase that dissociates Snare pairs

HIV enters through membrane fusionInfluenza enters through receptor-mediated endocytosis

Snare-like

IM-likeProteinsHydrophobicTails exposed