Synthesis and farnesylation of a-factor fusion proteins in Saccharomyces cerevisiae.
We have generated in-frame fusions between the mouse dihydrofolate reductase (DHFR) and parts of the a-factor MFA1 gene to explore the potential of a-factor as a secretion signal for larger polypeptides. We demonstrated that the fusion proteins are farnesylated by comparing the mobility of fusion proteins prepared from a wild-type strain and a farnesyltransferase mutant (ste16/ram1) on SDS-gels and by an in vitro farneyslation assay. In contrast to unmodified DHFR, the fusion proteins could be sedimented from cell extracts by centrifugation. Solubilization experiments indicated that the highly hydrophobic a-factor moiety renders the fusion proteins insoluble, explaining why the fusions are not secreted into the culture medium.
Kölling
R
R
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Böttner
B
B
Hollenberg
C P
CP
D000595:Amino Acid Sequence; D000818:Animals; D002498:Centrifugation; D000072235:Mating Factor; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor; D013762:Tetrahydrofolate Dehydrogenase