Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae.
Clathrin is important but not essential for yeast cell growth and protein secretion. Diploid Saccharomyces cerevisiae cells heterozygous for a clathrin heavy-chain gene (CHC1) disruption give rise to viable, slow-growing, clathrin heavy-chain-deficient meiotic progeny (G. Payne and R. Schekman, Science 230:1009-1014, 1985). The possibility that extragenic suppressors account for growth of clathrin-deficient cells was examined by deletion of CHC1 from haploid cell genomes by single-step gene transplacement and independently by introduction of a centromere plasmid carrying the complete CHC1 gene into diploid cells before eviction of a chromosomal CHC1 locus and subsequent tetrad analysis. Both approaches yielded clathrin-deficient haploid strains. In mutants missing at least 95% of the CHC1 coding domain, transcripts related to CHC1 were not detected. The time course of invertase modification and secretion was measured to assess secretory pathway functions in the viable clathrin-deficient cells. Core-glycosylated invertase was converted to the mature, highly glycosylated form at equivalent rates in mutant and wild-type cells. Export of mature invertase from mutant cells was delayed but not prevented. Abnormal vacuoles, accumulated vesicles, and Golgi body-derived structures were visualized in mutant cells by electron microscopy. We conclude that extragenic suppressors do not account for the viability of clathrin-deficient cells and, furthermore, that many standard laboratory strains can sustain a CHC1 disruption. Clathrin does not appear to mediate protein transfer from the endoplasmic reticulum to the Golgi body but may function at a later stage of the secretory pathway.
Payne
G S
GS
Department of Biochemistry, University of California, Berkeley 94720.
Hasson
T B
TB
Hasson
M S
MS
Schekman
R
R
D002966:Clathrin / Q000235:genetics*; D005796:Genes; D005800:Genes, Fungal; D005838:Genotype; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D013045:Species Specificity; D043324:beta-Fructofuranosidase
D002966:Clathrin; D046911:Macromolecular Substances; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase