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.

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 A late Golgi sorting function for Saccharomyces cerevisiae Apm1p, but not for Apm2p, a second yeast clathrin AP medium chain-related protein.

1995 ◽  
Vol 6 (1) ◽  
pp. 41-58 ◽  
Author(s):  
J D Stepp ◽  
A Pellicena-Palle ◽  
S Hamilton ◽  
T Kirchhausen ◽  
S K Lemmon
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Heavy Chain Gene ◽  
Medium Chain ◽  
Clathrin Heavy Chain ◽  
Sensitive Allele ◽  
Yeast Genes

Mammalian clathrin-associated protein (AP) complexes, AP-1 (trans-Golgi network) and AP-2 (plasma membrane), are composed of two large subunits of 91-107 kDa, one medium chain (mu) of 47-50 kDa and one small chain (sigma) of 17-19 kDa. Two yeast genes, APM1 and APM2, have been identified that encode proteins related to AP mu chains. APM1, whose sequence was reported previously, codes for a protein of 54 kDa that has greatest similarity to the mammalian 47-kDa mu 1 chain of AP-1. APM2 encodes an AP medium chain-related protein of 605 amino acids (predicted molecular weight of 70 kDa) that is only 30-33% identical to the other family members. In yeast containing a normal clathrin heavy chain gene (CHC1), disruptions of the APM genes, singly or in combination, had no detectable phenotypic consequences. However, deletion of APM1 greatly enhanced the temperature-sensitive growth phenotype and the alpha-factor processing defect displayed by cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. In contrast, deletion of APM2 caused no synthetic phenotypes with clathrin mutants. Biochemical analysis indicated that Apm1p and Apm2p are components of distinct high molecular weight complexes. Apm1p, Apm2p, and clathrin cofractionated in a discrete vesicle population, and the association of Apm1p with the vesicles was disrupted in CHC1 deletion strains. These results suggest that Apm1p is a component of an AP-1-like complex that participates with clathrin in sorting at the trans-Golgi in yeast. We propose that Apm2p represents a new class of AP-medium chain-related proteins that may be involved in a nonclathrin-mediated vesicular transport process in eukaryotic cells.

scholarly journals Fusion of the ALK Gene to the Clathrin Heavy Chain Gene, CLTC, in Inflammatory Myofibroblastic Tumor

2001 ◽  
Vol 159 (2) ◽  
pp. 411-415 ◽  
Author(s):  
Julia A. Bridge ◽  
Masahiko Kanamori ◽  
Zhigui Ma ◽  
Diane Pickering ◽  
D. Ashley Hill ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Inflammatory Myofibroblastic Tumor ◽  
Chain Gene ◽  
Heavy Chain Gene ◽  
Clathrin Heavy Chain

scholarly journals Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression.

1994 ◽  
Vol 126 (3) ◽  
pp. 689-699 ◽  
Author(s):  
R M Cripps ◽  
K D Becker ◽  
M Mardahl ◽  
W A Kronert ◽  
D Hodges ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Copy Number ◽  
Chain Gene ◽  
Wild Type ◽  
Heavy Chain Gene ◽  
Myosin Heavy Chain Gene ◽  
Flight Muscles ◽  
Mutant Phenotypes

We have transformed Drosophila melanogaster with a genomic construct containing the entire wild-type myosin heavy-chain gene, Mhc, together with approximately 9 kb of flanking DNA on each side. Three independent lines stably express myosin heavy-chain protein (MHC) at approximately wild-type levels. The MHC produced is functional since it rescues the mutant phenotypes of a number of different Mhc alleles: the amorphic allele Mhc1, the indirect flight muscle and jump muscle-specific amorphic allele Mhc10, and the hypomorphic allele Mhc2. We show that the Mhc2 mutation is due to the insertion of a transposable element in an intron of Mhc. Since a reduction in MHC in the indirect flight muscles alters the myosin/actin protein ratio and results in myofibrillar defects, we determined the effects of an increase in the effective copy number of Mhc. The presence of four copies of Mhc results in overabundance of the protein and a flightless phenotype. Electron microscopy reveals concomitant defects in the indirect flight muscles, with excess thick filaments at the periphery of the myofibrils. Further increases in copy number are lethal. These results demonstrate the usefulness and potential of the transgenic system to study myosin function in Drosophila. They also show that overexpression of wild-type protein in muscle may disrupt the function of not only the indirect flight but also other muscles of the organism.

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.

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.

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