CLATHRIN-DEPENDENT LOCALIZATION OF ALPHA-1,3 MANNOSYLTRANSFERASE TO THE GOLGI-COMPLEX OF SACCHAROMYCES-CEREVISIAE

Posttranslational modification of yeast glycoproteins with alpha 1,3-l inked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes. We have characterized th e synthesis, posttranslational modification, and localization of the y east alpha 1,3 mannosyltransferase (Mnn1p) using antibodies prepared a gainst a segment of this protein expressed in bacteria. Mnn1p is initi ally synthesized as a 98.5-kD, type II integral membrane glycoprotein that is modified with both N- and O-linked oligosaccharides. It is sub ject to a slow, incremental increase in molecular mass that is depende nt upon protein transport to the Golgi complex. Self-modification of M nn1p with alpha 1,3 mannose epitopes, primarily on O-linked oligosacch arides, is at least partly responsible for the incremental increase in molecular mass. Mnn1p is a resident protein of the Golgi complex and colocalizes with guanosine diphosphatase to at least two physically di stinct Golgi compartments by sucrose gradient fractionation, one of wh ich may be a late Golgi compartment that also contains the Kex2 endope ptidase. Surprisingly, we found that a significant fraction of Mnn1p i s mislocalized to the plasma membrane in a clathrin heavy chain temper ature sensitive mutant while guanosine diphosphatase remains intracell ular. A mutant Mnn1p that lacks the NH2-terminal cytoplasmic tail is p roperly localized to the Golgi complex, indicating that clathrin does not mediate Mnn1p Golgi retention by a direct interaction with the Mnn 1p cytoplasmic tail. These results indicate that clathrin plays a broa der role in the localization of Golgi proteins than anticipated.