LOCALIZATION OF A YEAST EARLY GOLGI MANNOSYLTRANSFERASE, OCH1P, INVOLVES RETROGRADE TRANSPORT

To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Oc h1p, a cis-Golgi mannosyltransferase. A series of influenza virus hema gglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-l ength Och1p, Several constructs included a Kex2p cleavage site between the Och1p and invertase moieties to monitor transit to the Kex2p-cont aining TGN. Cells expressing an Och1p-invertase fusion do not secrete invertase, but those expressing an Och1p-Kex2p site-invertase fusion. protein secrete high levels of invertase in a Kex2p-dependent manner. The Och1p-Kex2p site-invertase fusion protein is cleaved with a half-t ime of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage thro ugh the medial- and trans-Golgi, therefore cleavage occurs after order ed anterograde transport through the Golgi to the TGN. Transit to dist al compartments is not induced by the invertase moiety, since noninver tase fusion constructs encounter the same glycosyltransferases and Kex 2p as well. The Och1p-HA moiety, irrespective of whether it is generat ed by cleavage of the fusion protein in the TGN or synthesized de novo , is degraded with a half-time of about 60 min. Thus, the half-time of degradation is 12-fold longer than the time required to reach the TGN . At steady state, de novo-synthesized and TGN-generated HA epitope-ta gged Och1p reside in a compartment with a buoyant density identical to that of wild-type Och1p and distinct from that of the vacuole or the TGN. Finally, och1 null cells that express an Och1p fusion construct k nown to rapidly encounter the TGN glycosylate invertase to the same ex tent as wild-type cells, indicating that they have phenotypically wild -type Och1p activity. These results lead us to propose a model for Och 1p-HA localization that involves movement to distal compartments, at l east as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.