THE CYTOPLASMIC TAIL OF MOUSE HEPATITIS-VIRUS M-PROTEIN IS ESSENTIAL BUT NOT SUFFICIENT FOR ITS RETENTION IN THE GOLGI-COMPLEX

The M protein of mouse hepatitis virus (MHV) is a triple-spanning memb rane glycoprotein that is exclusively O-glycosylated. When expressed i ndependently, it accumulates in late Gels and the trans-Golgi network (TGN) (Locker, J. K., Griffiths, G., Horzinek, M. C., and Rottier, P. J. M. (1992) (J. Biol. Chem. 267, 14094-14101). To analyze the domains of this protein responsible for its localization, we have generated d eletion mutants by site-directed mutagenesis and analyzed their intrac ellular transport. The intracellular distribution of the mutant protei ns was determined by following the extent of O-glycosylation in pulse- chase experiments, by electron microscopic immunocytochemistry, and by surface immunoprecipitation. Mutant proteins lacking the first or the first and second transmembrane domains were not efficiently retained in the Golgi complex or TGN. The latter mutant proteins also localized to endocytic compartments but were not subject to rapid lysosomal deg radation. Deletion of the COOH-terminal 22 amino acids, including a ty rosine residue in the context of a potential internalization signal, r esulted in plasma membrane exposure of the respective mutant protein, We show that the wild-type MHV-M protein does not recycle between the plasma membrane and the TGN, but rather behaves as a late Golgi/TGN re sident in our assays. We propose that the MHV-M protein is retained in the Golgi by two signals, one contained in the cytoplasmic tail and t he other determined by the transmembrane domains.