Yeast Genes Controlling Responses to Topogenic Signals in a Model Transmembrane Protein

Abstract

Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein. Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2–3% by a strong charge difference signal. Identifiedpio mutants increased this to 9–12%.PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi.spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca²⁺ATPase and Spf1p may be the equivalent ER pump.PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects ofste24- and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1- orste24-null mutants. Mutations in PIO3(unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins.