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The M protein of mouse hepatitis virus (MHV) is a triple-spanning membrane glycoprotein that is exclusively O-glycosylated. When expressed independently, it accumulates in late Golgi 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 deletion mutants by site-directed mutagenesis and analyzed their intracellular transport. The intracellular distribution of the mutant proteins 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 degradation. Deletion of the COOH-terminal 22 amino acids, including a tyrosine residue in the context of a potential internalization signal, resulted 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 resident 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 the other determined by the transmembrane domains.

The membrane insertion of the E1 protein of a coronavirus, mouse hepatitis virus A59, was studied in a wheat germ cell-free translation system. E1 is a transmembrane protein spanning the lipid bilayer several times. It is synthesized without a cleavable signal sequence, localized intracellularly, and not transported to the cell surface. It thus represents a model intracellular protein. We found that the synthesis of E1 is specifically and stably blocked by the addition of signal recognition particle to the wheat germ system. Subsequent addition of salt-extracted pancreatic microsomes resulted in the full release of this arrest as well as the completion and the correct membrane integration of E1. Such signal recognition particle-induced arrests failed to produce shorter peptides of a defined length. Addition of signal recognition particle to a synchronized translation at any time during the synthesis of about the first two thirds of E1 (150 amino acids) blocked further translation, suggesting that the most C-terminal of the three internal hydrophobic domains of E1 could function as its signal sequence.