Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/ 24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in S accharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodi mer. We found that cytosol from an lst1-null strain supported the packaging of or-factor precursor into COPII vesicles but was deficient in the packag ing of Pma1p, the essential plasma membrane ATPase. Supplementation of muta nt cytosol with purified Scc23/Lst1p restored Pma1p packaging into the vesi cles. When purified COPII components were used in the vesicle budding react ion, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1 p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecip itated with Lst1p and Sec24p from vesicles, Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoy ant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diamete r). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/L st1p and Sec23/24p on the membranes of the same vesicles. These results sug gest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be s orted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geom etry of COPII coat polymerization, Lst1p may allow the transport of bulky c argo molecules, polymers, or particles.