Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor.
The proteolytic processes involved in the cotranslational production of the Semliki Forest virus proteins p62, 6K, and E1 from a common precursor polypeptide were analyzed by an in vitro translation-translocation assay. By studying the behavior of wild-type and mutant variants of the polyprotein, we show that the signal sequences responsible for membrane translocation of the 6K and E1 proteins reside in the C-terminal regions of p62 and 6K, respectively. We present evidence suggesting that the polyprotein is processed on the luminal side by signal peptidase at consensus cleavage sites immediately following the signal sequences. Our results also lead us to conclude that the 6K protein is a transmembrane polypeptide with its N terminus on the luminal side of the membrane (type I). Thus, the production of all three membrane proteins is directed by alternating signal and stop-transfer (anchor) sequences that function in translocation and cleavage of the virus precursor polyprotein. This also shows conclusively that internally located signal sequences can be cleaved by signal peptidase.
Liljeström
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P
Department of Molecular Biology, Karolinska Institute, Huddinge, Sweden.
Garoff
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H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*; D014763:Viral Matrix Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D021382:Protein Sorting Signals; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins