Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport

The yeast SNARE Ykt6p has been implicated in several trafficking steps, inc luding vesicular transport from the endoplasmic reticulum (ER) to the Golgi , intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homologue (Ykt6) has not been established. Using antibodie s specific for mammalian Ykt6, it is revealed that it is found mainly in Go lgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are speci fically associated with Ykt6 as revealed by co-immunoprecipitation, suggest ing that these four SNAREs form a SNARE complex. Double labeling of Ykt6 an d the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL rec eptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exi t sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of ves icular stomatitis virus envelope glycoprotein (VSVG) only when they are add ed before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Y kt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented f rom entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosida se II is fragmented into vesicular structures in cells microinjected with Y kt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Go lgi transport, and this role may be important for the integrity of the Golg i complex.