SCD5, a suppressor of clathrin deficiency, encodes a novel protein with a late secretory function in yeast.

Abstract

Clathrin and its associated proteins constitute a major class of coat proteins involved in vesicle budding during membrane transport. An interesting characteristic of the yeast clathrin heavy chain gene (CHC1) is that in some strains a CHC1 deletion is lethal, while in others it is not. Recently, our laboratory developed a screen that identified five multicopy suppressors that can rescue lethal strains of clathrin heavy chain-deficient yeast (Chc - scd1-i) to viability. One of these suppressors, SCD5, encodes a novel protein of 872 amino acids containing two regions of repeated motifs of unknown function. Deletion of SCD5 has shown that it is essential for cell growth at 30 degrees C. scd5-delta strains carrying low copy plasmids encoding C-terminal truncations of Scd5p are temperature sensitive for growth at 37 degrees C. At the nonpermissive temperature, cells expressing a 338-amino acid deletion (Scd5P-delta 338) accumulate an internal pool of fully glycosylated invertase and mature alpha-factor, while processing and sorting of the vacuolar hydrolase carboxypeptidase Y is normal. The truncation mutant also accumulates 80- to 100-nm vesicles similar to many late sec mutants. Moreover, at 34 degrees C, overexpression of Scd5p suppresses the temperature sensitivity of a sec2 mutant, which is blocked at a post-Golgi step of the secretory pathway. Biochemical analyses indicate that approximately 50% of Scd5p sediments with a 100,000 x g membrane fraction and is associated as a peripheral membrane protein. Overall, these results indicate that Scd5p is involved in vesicular transport at a late stage of the secretory pathway. Furthermore, this suggests that the lethality of clathrin-deficient yeast can be rescued by modulation of vesicular transport at this late secretory step.

Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis

“…We identified Scd5 in a screen for multicopy suppressors of clathrin deficiency, and later showed its importance in endocytosis and cortical actin organization Nelson et al, 1996). Binding of Glc7 to the canonical [R/K]x 0-1 [V/I]xF PP1 binding motif of Scd5 (Egloff et al, 1997) is critical for the endocytic function of Scd5 .…”

The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

“…These include periplasmic glycoproteins such as invertase (Schekman and Novick, 1982), proteins secreted into the extracellular medium such as the pheromone a-factor (Julius et al, 1984;Nelson et al, 1996), and plasma membrane permeases (Novick and Schekman, 1979;Tschopp et al, 1984) including uracil permease, which we use in this study. Uracil permease, encoded by the FUR4 gene (Jund et al, 1988), is targeted to the plasma membrane where it undergoes phosphorylation on multiple serine residues (Volland et al, 1992).…”

Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

“…These gene products are encoded by SECI, SEC2, SEC3, SEC4, SEC5, SEC6, SEC8, SEC9, SEC10, SEC15, SSO1, SS02, SNC1, SNC2, and SCD5 (Novick and Schekman, 1979;Novick et al, 1980;Gerst et al, 1992;Aalto et al, 1993;Protopopov et al, 1993;Nelson et al, 1996). All stages of vesicular transport require members of particular protein families, termed rabs and SNAREs.…”