Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum

Mnn9p is a component of two distinct multiprotein complexes in the Saccharo myces cerevisiae cis-Golgi that have both been shown to have alpha -1,6-man nosyltransferase activity in vitro. In one of these complexes, Mnn9p associ ates with four other membrane proteins. Anp1p. Mnn10p, Mnn11p, and Hoc1p, w hereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p- containing complexes were incorporated into COPII Vesicles made in vitro fr om endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells. This behavior is consistent with an active Golgi to ER recycling pr ocess. To examine this path in vivo, we monitored retrograde transport of s ubunits of the complex in cells blocked in anterograde transport from the E R. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, w hen retrograde transport was blocked in vivo, subunits of the mannosyltrans ferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coate d Vesicles from purified Golgi membranes was also investigated using a coat omer-dependent Vesicle budding assay. Gradient fractionation experiments sh owed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golg i.