Membrane integration and intracellular transport of the coronavirus glycoprotein E1, a class III membrane glycoprotein.
The E1-glycoprotein (Mr = 26,014; 228 amino acids) of mouse hepatitis virus A59 is a class III membrane glycoprotein which has been used in this study as a model system in the study of membrane integration and protein transport. The protein lacks an NH2-terminal cleavable signal sequence and spans the viral membrane three times. Hydrophobic domains I and III could serve as signal sequences for cotranslational membrane integration. Domain I alone was sufficient to translocate the hydrophilic NH2 terminus of E1 across the membranes as evidenced by glycosylation of a newly introduced N-glycosylation site. The COOH-terminal part of E1 involving amino acids Leu124 to Thr228 was found to associate tightly with membranes at the post-translational level, although this part of the molecule lacks pronounced hydrophobic sequences. Membrane protection assays with proteinase K showed that a 2-kDa hydrophilic fragment was removed from the COOH terminus of E1 indicating that the protein is largely embedded into the membrane. Microinjection of in vitro transcribed capped and polyadenylated mRNA into CV-1 cells or into secretory AtT20 pituitary tumor cells showed that the E1-protein accumulated in the Golgi but was not detectable at the plasma membrane or in secretory granules. The 28 NH2-terminal hydrophilic amino acid residues play no role in membrane assembly or in intracellular targeting. Various NH2-terminal portions of E1 were fused to Ile145 of the cytoplasmic N-protein of mouse hepatitis virus. The resulting hybrid proteins were shown to assemble into membranes in vitro and were detected either in the rough endoplasmic reticulum or transient vesicles of microinjected cells.
Mayer
T
T
Institut für Medizinische Virologie, Justus-Liebig-Universität, Giessen, Federal Republic of Germany.
Tamura
T
T
Falk
M
M
Niemann
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins