Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport.

Abstract

To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Och1p, a cis-Golgi mannosyltransferase. A series of influenza virus hemagglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-length Och1p. Several constructs included a Kex2p cleavage site between the Och1p and invertase moieties to monitor transit to the Kex2p-containing 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-time of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage through the medial- and trans-Golgi, therefore cleavage occurs after ordered anterograde transport through the Golgi to the TGN. Transit to distal compartments is not induced by the invertase moiety, since noninvertase fusion constructs encounter the same glycosyltransferases and Kex2p as well. The Och1p-HA moiety, irrespective of whether it is generated 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-tagged 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 Ochlp fusion construct known to rapidly encounter the TGN glycosylate invertase to the same extent as wild-type cells, indicating that they have phenotypically wild-type Och1p activity. These results lead us to propose a model for Och1p-HA localization that involves movement to distal compartments, at least as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

“…Vps53p-HA and Vps54p-HA exhibited a similar staining pattern (our unpublished results), which is typical of many Golgi membrane proteins (Redding et al, 1991;Nothwehr et al, 1993;Cooper and Stevens, 1996). In S. cerevisiae, the Golgi complex is not organized into stacked cisternae, and components of early and late Golgi compartments can readily be distinguished by immunofluorescence microscopy (Harris and Waters, 1996;Holthuis et al, 1998a). Most of the structures that contained Vps52p-HA were also found to label with the late Golgi marker A-ALP, although there were some differences in the relative intensities of the structures labeled by each antibody ( Figure 10A).…”

“…Ura ϩ transformants were plated on 5-FOA, and those cells expressing Vps10p-HA were identified by Western blotting and tested for CPY sorting. To integrate the Och1p-HA-encoding allele, cells were transformed with EcoRI-cut pLC116, which contains the MfeIHindIII fragment from pOH (Harris and Waters, 1996) subcloned into the EcoRI-HindIII sites of pRS306.…”

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

“…Alpha 1,2-mannosidase activity was separated in two peaks, with 32% of the activity associated with densities in the range reported for Golgi (1.122-1.142 g cm -3 ) and 68% of the activity associated with densities reported for ER membranes (1.178-1.182 g cm -3 ) (Beaufay et al 1974, Chrispeels et al 1982, Harris & Waters 1996, Mora-Montes et al 2008a, consistent with distribution of the enzyme between the Golgi complex and ER. Monensin blocks the vesicle transport within the Golgi complex (Rosa et al 1992) and, as a consequence, proteins are accumulated in the ER.…”

Mutations in a Highly Conserved Region of the Arf1p ActivatorGEA2Block Anterograde Golgi Transport but Not COPI Recruitment to Membranes

“…Sec7-GFP structures also decreased in number after shift to the nonpermissive temperature. On the other hand, structures bearing the early Golgi markers Och1p-HA (Harris and Waters, 1996) and Sec21p-GFP (Rossanese et al, 2001) were still present after shift to the nonpermissive temperature (Figure 4). Sec21p-GFP was present in a pattern of spots in all four mutants even after shift to the nonpermissive temperature for 40 min (Figure 4 and our unpublished data).…”