1. Molecular mechanism of vesicular transport Vesicular pathway--Budding and fusion 1.Budding— how are vesicles formed? How can cargo proteins be selectively packaged into different vesicles? 1)At least 3 types of coated vesicles transport proteins to different places 2) Clathrin coated vesicles (CCV): typical CCV is 50-100 nm in diameter with a membrane vesicle inside the coat made of fibrous protein clathrin. Purified clathrin has three limbs called triskelions (Greek 3 legs). Each leg is made of a heavy chain (180kD) and a light chain (40kD). The triskelion can polymerize to form a polygonal cage surrounding the vesicles.

2. The 3 types of coats we want to focus on are clathrin, COP1 and COPII. CCV mediates Golgi-Vacuole traffic, COPI mediates retrograde traffic from Golgi to ER; COPII mediate ER—Golgi transport Obviously, other coats are there to be identified because these 3 types do not cover all vesicles.

3. Clathrin “pits” in the membrane where budding is initiated. The triskelion structure of clathrin

4. Formation of clathrin triskelion and polygonal basket. The adaptor proteins provide the link between clathrin and cargo receptors.

5. 3) Clathrin interaction with other components in the budding sites: --the adaptor proteins (AP): at least three types of APs have been identified AP1, AP2, and AP3 that mediate clathrin interaction with cargo receptors that in turn specify cargo proteins—which proteins to be packaged. For example, the M-6-P receptors will pack the M-6-P proteins destined to the vacuole/lysosome. The M-6-P receptors interact with AP proteins that interact with clathrin coat. Soluble cargo Receptor AP Clathrin coat

6. 4) The budding process: The cargo binding to receptor and integral proteins interaction with adaptor proteins trigger interaction with clathrin proteins that spontaneously form the coat structure. Formation of coat structure initiates the “budding”---budged membrane forms a “bump”. At the neck of the bump, a protein called dynamin, a GTP-binding protein (GTPase), that polymerizes and help the bud pinch off the membrane. 5) Coat Shedding of the CCV: CCV rapidly shed its coat after vesicle formation. This process requires a chaperone hsp70, a ATPase, that use ATP hydrolysis to depolymerize the clathrin coat into the triskelion subunits—to be re-used in the budding process.

8. 6) The formation of COPI vesicles: Coatomer proteins instead of clathrin cover many of the vesicles formed from Golgi going to the ER. These proteins (many different subunits), like clathrin, also polymerize into a cage-like structure that surrounds the vesicle. A small GTP-binding protein called ARF initiates this budding process. ARF receptor and other integral membrane proteins interact with the COPI subunits. 7) Dissociation of COPI coat from the vesicles is triggered by the GTP hydrolysis of ARF. If you inhibit this hydrolysis, the coat will not be shed. COPI vesicles are involved in Golgi-ER trafficking: study of yeast secretion mutants. If KDEL receptor mutated or COPI subunits are mutated, the ER proteins can not go back to ER—stuck into the cis-Golgi. Yeast genetics provides a powerful tool for studying the protein sorting.

9. 8) Formation of COPII vesicles are similar to COPI: Different coatomer proteins that are related to COPI coatomers. The initiation of budding is also triggered by a small G protein called SAR1 related to ARF in structure. Yeast mutants lacking some of these components will accumulate proteins in the ER—so COPII is involved in ER-Golgi trafficking… 2. Fusion of vesicles to the target membrane Questions: what is the signal that tells The vesicle to selectively fuse to the Correct target? How does a vesicle Fuse to the target membrane?

10. 1)SNAREs: that hold the key for vesicle and target recognition a)Before fusion, the coats of the vesicles must shed. b) SNARE proteins were exposed after the coats are dissociated. Both vesicle and target membrane has SNAREs. Those on the vesicle are called v-SNAREs, and those on the target membrane are called t-SNAREs. The v-SNARE and t-SNARE pair up with each other to specify which vesicle fuse with which target membrane. c) SNAREs are sufficient for fusion to occur: Biochemical studies: liposomes containing the v- and t-SNAREs can fusion selectively in vitro. Genetics: yeast mutants in SNAREs had trouble fusing vesicles with target… d) Small GTP binding protein Rab is essential for efficient fusion of the vesicles to the membrane. e) Names: NSF, SNAP, and SNARE: first a NEM-sensitive factor (SNF) was found to be involved in fusion, then a SNF- Attachment Protein (SNAP) was found, then came the SNAP REceptor in the membrane (SNARE)

11. V-SNARE T-SNARE Rab

12. 2) The protein pore model: to suggest that SNARE protein pairs interact and form “pore” or channel-like structure that initiates that fusion process so that the vesicle contents are not “spilled”. Summary: Coat is to specify cargo and SNARE is to match the cargo with the location.

13. 3. What is known about vesicular trafficking plant cells Most of work has been done on vacuole protein targeting using storage/lytic vacuole proteins as models. 1) Three types of trafficking mechanisms exist for vacuole targeting

14. 2) Vacuole targeting depends on presence of signal sequence in the cargo proteins. Two major signals are identified: N-terminal and C- terminal regions. Some also found internal sequences that are critical for targeting. It is complex

15. 3) A number of SNAREs have been identified in plant genomes. Some work has shown that they are important for growth and development and signaling. 4) PI metabolism may be involved in secretion pathway, like in animal/yeast. For example, a phosphotidylinositol 3-kinase appears to be important for vacuole targeting.