scholarly journals The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1119-1134 ◽  
Author(s):  
C Bazinet ◽  
A L Katzen ◽  
M Morgan ◽  
A P Mahowald ◽  
S K Lemmon
Keyword(s):  
X Chromosome ◽  
Heavy Chain ◽  
P Element ◽  
Cdna Clones ◽  
Drosophila Genome ◽  
Wild Type ◽  
Degenerate Primers ◽  
Coated Pits ◽  
Lethal Mutations ◽  
Clathrin Heavy Chain

Abstract The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis.

scholarly journals Elimination of Introns at the Drosophila suppressor-of-forked Locus by P-Element-Mediated Gene Conversion Shows That an RNA Lacking a Stop Codon is Dispensable

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 345-351
Author(s):  
Carol J Williams ◽  
Kevin O'Hare
Keyword(s):  
X Chromosome ◽  
Stop Codon ◽  
Alternative Polyadenylation ◽  
Gene Replacement ◽  
P Element ◽  
Wild Type ◽  
Genomic Location ◽  
Chromosomal Breaks ◽  
White Locus ◽  
Cleavage Stimulation Factor

Abstract The suppressor of forked [su(f)] locus affects the phenotype of mutations caused by transposable element insertions at unlinked loci. It encodes a putative 84-kD protein with homology to two proteins involved in mRNA 3′ end processing; the product of the yeast RNA14 gene and the 77-kD subunit of human cleavage stimulation factor. Three su(f) mRNAs are produced by alternative polyadenylation. The 2. 6 and 2.9-kb mRNAs encode the same 84-kD protein while a 1.3-kb RNA, which terminates within the fourth intron, is unusual in having no stop codon. Using P-element-mediated gene replacement we have copied sequences from a transformation construct into the su(f) gene creating a su(f) allele at the normal genomic location that lacks the first five introns. This allele is viable and appears wild type for su(f) function, demonstrating that the 1.3-kb RNA and the sequences contained within the deleted introns are dispensable for su(f) function. Compared with studies on gene replacement at the white locus, chromosomal breaks at su(f) appear to be less efficiently repaired from ectopic sites, perhaps because of the location of su(f) at the euchromatin/heterochromatin boundary on the X chromosome.

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 Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

2003 ◽  
Vol 163 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Antony P. Jackson ◽  
Alexander Flett ◽  
Carl Smythe ◽  
Lindsay Hufton ◽  
Frank R. Wettey ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Transferrin Receptor ◽  
Coated Pits ◽  
Permeabilized Cells ◽  
Clathrin Heavy Chain

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

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.

Internalization of cholera toxin by different endocytic mechanisms

2001 ◽  
Vol 114 (20) ◽  
pp. 3737-3747 ◽  
Author(s):  
Maria L. Torgersen ◽  
Grethe Skretting ◽  
Bo van Deurs ◽  
Kirsten Sandvig
Keyword(s):  
Cholera Toxin ◽  
Heavy Chain ◽  
Large Fraction ◽  
Cell Types ◽  
Coated Pits ◽  
Bhk Cells ◽  
Clathrin Heavy Chain ◽  
Antisense Mrna ◽  
Inducible Synthesis ◽  
Different Cell Types

The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-β-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.

scholarly journals The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

1996 ◽  
Vol 7 (11) ◽  
pp. 1667-1677 ◽  
Author(s):  
K Redding ◽  
M Seeger ◽  
G S Payne ◽  
R S Fuller
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Heavy Chain ◽  
Intracellular Transport ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Localization Signal ◽  
Mutant Forms ◽  
Kex2 Protease

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.

scholarly journals Zygotic Lethal Mutations With Maternal Effect Phenotypes in Drosophila melanogaster. II. Loci on the Second and Third Chromosomes Identified by P-Element-Induced Mutations

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1681-1692 ◽  
Author(s):  
Norbert Perrimon ◽  
Anne Lanjuin ◽  
Charles Arnold ◽  
Elizabeth Noll
Keyword(s):  
Maternal Effect ◽  
Genome Project ◽  
P Element ◽  
Berkeley Drosophila Genome Project ◽  
Drosophila Genome ◽  
Gene Products ◽  
Induced Mutations ◽  
Lethal Mutations ◽  
Egg Formation ◽  
Normal Embryonic Development

Screens for zygotic lethal mutations that are associated with specific maternal effect lethal phenotypes have only been conducted for the X chromosome. To identify loci on the autosomes, which represent four-fifths of the Drosophila genome, we have used the autosomal “FLP-DFS” technique to screen a collection of 496 P element-induced mutations established by the Berkeley Drosophila Genome Project. We have identified 64 new loci whose gene products are required for proper egg formation or normal embryonic development.

scholarly journals Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium.

1992 ◽  
Vol 118 (6) ◽  
pp. 1371-1377 ◽  
Author(s):  
T J O'Halloran ◽  
R G Anderson
Keyword(s):  
Heavy Chain ◽  
Antisense Rna ◽  
Fluid Phase ◽  
Solid Particles ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Clathrin Heavy Chain ◽  
Development Cycle ◽  
Mutant Cells

To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.

A P-Element Insertion Screen Identified Mutations in 455 Novel Essential Genes in Drosophila

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 195-201 ◽  
Author(s):  
Su-Wan Oh ◽  
Tracy Kingsley ◽  
Hyun-hee Shin ◽  
Zhiyu Zheng ◽  
Hua-Wei Chen ◽  
...  
Keyword(s):  
Biological Function ◽  
P Element ◽  
Drosophila Genome ◽  
Gene Products ◽  
Induced Mutations ◽  
Element Insertion ◽  
Lethal Mutations ◽  
Chromosome Mutations ◽  
Eukaryotic Genomes ◽  
Insertion Mutations

Abstract With the completion of the nucleotide sequences of several complex eukaryotic genomes, tens of thousands of genes have been predicted. However, this information has to be correlated with the functions of those genes to enhance our understanding of biology and to improve human health care. The Drosophila transposon P-element-induced mutations are very useful for directly connecting gene products to their biological function. We designed an efficient transposon P-element-mediated gene disruption procedure and performed genetic screening for single P-element insertion mutations, enabling us to recover 2500 lethal mutations. Among these, 2355 are second chromosome mutations. Sequences flanking >2300 insertions that identify 850 different genes or ESTs (783 genes on the second chromosome and 67 genes on the third chromosome) have been determined. Among these, 455 correspond to genes for which no lethal mutation has yet been reported. The Drosophila genome is thought to contain ∼3600 vital genes; 1400 are localized on the second chromosome. Our mutation collection represents ∼56% of the second chromosome vital genes and ∼24% of the total vital Drosophila genes.

scholarly journals Studies on the rate and site-specificity of P element transposition.

Genetics ◽  
1991 ◽  
Vol 127 (3) ◽  
pp. 515-524 ◽  
Author(s):  
C A Berg ◽  
A C Spradling
Keyword(s):  
X Chromosome ◽  
Chromosomal Location ◽  
The Other ◽  
P Element ◽  
Site Specificity ◽  
Drosophila Genome ◽  
P Elements ◽  
Genomic Location ◽  
Target Sites ◽  
And Function

Abstract A single genetically marked P element can be efficiently mobilized to insertionally mutagenize the Drosophila genome. We have investigated how the structure of the starting element and its location along the X chromosome influenced the rate and location of mutations recovered. The structure of two P[rosy+] elements strongly affected mobilization by the autonomous "Jumpstarter-1" element. Their average transposition rates differed more than 12-fold, while their initial chromosomal location had a smaller effect. The lethal and sterile mutations induced by mobilizing a P[rosy+] element from position 1F were compared with those identified previously using a P[neoR] element at position 9C. With one possible exception, insertion hotspots for one element were frequently also targets of the other transposon. These experiments suggested that the genomic location of a P element does not usually influence its target sites on nonhomologous chromosomes. During the course of these experiments, Y-linked insertions expressing rosy+ were recovered, suggesting that marked P elements can sometimes insert and function at heterochromatic sites.

Sign in / Sign up

Export Citation Format

Share Document