Clathrin-dependent localization of α1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae

Abstract

Posttranslational modification of yeast glycoproteins with α1,3-linked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes. We have characterized the synthesis, posttranslational modification, and localization of the yeast α1,3 mannosyltransferase (Mnn1p) using antibodies prepared against a segment of this protein expressed in bacteria. Mnn1p is initially synthesized as a 98.5- kD, type II integral membrane glycoprotein that is modified with both N- and O-linked oligosaccharides. It is subject to a slow, incremental increase in molecular mass that is dependent upon protein transport to the Golgi complex. Self-modification of Mnn1p with α1,3 mannose epitopes, primarily on O- linked oligosaccharides, 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 distinct Golgi compartments by sucrose gradient fractionation, one of which may be a late Golgi compartment that also contains the Kex2 endopeptidase. Surprisingly, we found that a significant fraction of Mnn1p is mislocalized to the plasma membrane in a clathrin heavy chain temperature sensitive mutant while guanosine diphosphatase remains intracellular. A mutant Mnn1p that lacks the NH2-terminal cytoplasmic tail is properly localized to the Golgi complex, indicating that clathrin does not mediate Mnn1p Golgi retention by a direct interaction with the Mnn1p cytoplasmic tail. These results indicate that clathrin plays a broader role in the localization of Golgi proteins than anticipated.