The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles

Sec2p is required for the polarized transport of secretory vesicles in S. c erevisiae. The Sec2p NH, terminus encodes an exchange factor for the Rab pr otein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal muta nts Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p CO OH terminus functions in targeting vesicles, however, the mechanism of func tion is unknown. We found comparable exchange activity for truncated and fu ll-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips, A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Se c6p, and Sec9p function. Full-length, but not COOH-terminally truncated Sec 2 proteins are enriched on membranes. Membrane association of full-length S ec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffe cted at 25 degrees C. Taken together, these data correlate loss of localiza tion of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the not ion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4 p.