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. Identified pio 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 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca 2ϩ 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 of ste24-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-or ste24-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.

The Sensitivity of Yeast Mutants to Oleic Acid Implicates the Peroxisome and Other Processes in Membrane Function

“…Deletion of PEX3, PEX6, or PEX15, as anticipated, allows little if any Pex11-GFP to accumulate even after oleate treatment. Spf1, identified in the oleate screen, is reported to be involved in ER function, calcium homeostasis (Cronin et al 2002), and the orientation of protein insertion into membranes (Tipper and Harley 2002). It was also shown by mass spectrometry to be associated with the peroxisome (Marelli et al 2004).…”

ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability

“…Cod1p, the PDR2/MIA ortholog in yeast (S. cerevisiae), is located in the ER and nuclear envelope (32). Properties of cod1⌬ strains point to its function in protein biogenesis and ER quality control, including protein processing, control of protein insertion orientation, ERAD, and cation homeostasis (33)(34)(35)(36). Several observations support a role for PDR2/MIA in the ER: (i) its C-terminal ER retrieval signal of the KKXX-type suggests retrograde Golgi-to-ER transport (37); (ii) a proteomics approach identified the At5g23630 protein in ER microsomes (21); and (iii) Jakobsen et al (20) detected MIA by immunogold labeling in the ER and small vesicles in tapetal cells and demonstrated its ability to complement cod1⌬.…”

Pollen development and fertilization in Arabidopsis is dependent on the MALE GAMETOGENESIS IMPAIRED ANTHERS gene encoding a Type V P-type ATPase

“…Spf1p has been localized to both the ER (Cronin et al 2002;Vashist et al 2002) and the cis-Golgi (Suzuki 2001). The protein functions in a variety of processes linked to protein processing such as N-glycosylation (Suzuki and Shimma 1999), control of hydroxyl-methylglutaryl-coenzyme A reductase 2 degradation (Cronin et al 2000), and control of protein insertion orientation (Tipper and Harley 2002). Furthermore, the expression of SPF1 is up-regulated by the unfolded protein response (Travers et al 2000), and spf1 is dependent on the activation of the unfolded protein response for viability (Ng et al 2000;Vashist et al 2002).…”