Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae

1987 ◽  
Vol 7 (11) ◽  
pp. 3888-3898
Author(s):  
G S Payne ◽  
T B Hasson ◽  
M S Hasson ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Secretory Pathway ◽  
Biochemical Characterization ◽  
Golgi Body ◽  
Chain Gene ◽  
Clathrin Heavy Chain ◽  
Diploid Cells ◽  
Extragenic Suppressors ◽  
Mutant Cells

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.

scholarly journals Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (11) ◽  
pp. 3888-3898 ◽  
Author(s):  
G S Payne ◽  
T B Hasson ◽  
M S Hasson ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Secretory Pathway ◽  
Biochemical Characterization ◽  
Golgi Body ◽  
Chain Gene ◽  
Clathrin Heavy Chain ◽  
Diploid Cells ◽  
Extragenic Suppressors ◽  
Mutant Cells

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.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

1991 ◽  
Vol 11 (8) ◽  
pp. 3868-3878 ◽  
Author(s):  
A L Munn ◽  
L Silveira ◽  
M Elgort ◽  
G S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Heavy Chain ◽  
Secretory Pathway ◽  
Genetic Locus ◽  
Wild Type ◽  
Site Mutation ◽  
Gene Encoding ◽  
Clathrin Heavy Chain ◽  
Lethal Allele

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.

scholarly journals Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (1) ◽  
pp. 521-532 ◽  
Author(s):  
K K Nelson ◽  
S K Lemmon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Genomic Library ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Selection Strategy ◽  
Chain Gene ◽  
Mrna Levels ◽  
General Role ◽  
Clathrin Heavy Chain

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive. With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable. To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed. A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected. Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.

scholarly journals Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function.

1991 ◽  
Vol 112 (1) ◽  
pp. 65-80 ◽  
Author(s):  
S K Lemmon ◽  
A Pellicena-Palle ◽  
K Conley ◽  
C L Freund
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Heavy Chain ◽  
Coiled Coil ◽  
Coated Vesicle ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Clathrin Heavy Chain ◽  
And Function ◽  
Major Defect

The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.

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

1996 ◽  
Vol 7 (2) ◽  
pp. 245-260 ◽  
Author(s):  
K K Nelson ◽  
M Holmer ◽  
S K Lemmon
Keyword(s):  
Heavy Chain ◽  
Secretory Pathway ◽  
Vesicular Transport ◽  
Carboxypeptidase Y ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Coat Proteins ◽  
Clathrin Heavy Chain ◽  
Biochemical Analyses ◽  
Novel Protein

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.

scholarly journals Genetic instability of clathrin-deficient strains of Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 124 (1) ◽  
pp. 27-38
Author(s):  
S K Lemmon ◽  
C Freund ◽  
K Conley ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Copy Number ◽  
Genetic Background ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Genome Copy ◽  
Clathrin Heavy Chain ◽  
Genome Copy Number ◽  
Genome Content

Abstract Saccharomyces cerevisiae strains carrying a mutation in the clathrin heavy chain gene (CHC1) are genetically unstable and give rise to heterogeneous populations of cells. Manifestations of the instability include increases in genome copy number as well as compensatory genetic changes that allow better growing clathrin-deficient cells to take over the population. Increases in genome copy number appear to result from changes in ploidy as well as alterations in normal nuclear number. Genetic background influences the frequency at which cells with increased genome content are observed in different Chc- strains. We cannot distinguish whether genetic background affects the rate at which aberrant nuclear division events occur or a growth advantage of cells with increased nuclear and/or genome content. However, survival of chc1-delta cells does not require an increase in genome copy number. The clathrin heavy chain gene was mapped 1-2 cM distal to KEX1 on the left arm of chromosome VII by making use of integrated 2 mu plasmid sequences to destabilize distal chromosome segments and allow ordering of the genes.

Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 521-532
Author(s):  
K K Nelson ◽  
S K Lemmon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Genomic Library ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Selection Strategy ◽  
Chain Gene ◽  
Mrna Levels ◽  
General Role ◽  
Clathrin Heavy Chain

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive. With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable. To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed. A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected. Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.

Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis

1991 ◽  
Vol 11 (8) ◽  
pp. 3868-3878
Author(s):  
A L Munn ◽  
L Silveira ◽  
M Elgort ◽  
G S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Heavy Chain ◽  
Secretory Pathway ◽  
Genetic Locus ◽  
Wild Type ◽  
Site Mutation ◽  
Gene Encoding ◽  
Clathrin Heavy Chain ◽  
Lethal Allele

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.

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