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1.
Mol Biol Cell ; 12(6): 1885-96, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11408593

ABSTRACT

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the "ear" domain of the clathrin adaptor AP-1 gamma subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Delta), the major Gga protein, accentuates growth and alpha-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Delta or a deletion of the AP-1 beta subunit gene (apl2Delta) alone are phenotypically normal, but cells carrying both gga2Delta and apl2Delta are defective in growth, alpha-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.


Subject(s)
ADP-Ribosylation Factors/chemistry , Carrier Proteins/chemistry , Clathrin/metabolism , Endosomes/metabolism , Golgi Apparatus/metabolism , ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Vesicular Transport , Carrier Proteins/metabolism , Cell Division , Chromatography, Affinity , Fungal Proteins/metabolism , Gene Deletion , Genotype , Glutathione Transferase/metabolism , Heterozygote , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Biological , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Subcellular Fractions , Temperature , Time Factors , trans-Golgi Network/metabolism
2.
Mol Biol Cell ; 12(1): 13-26, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11160819

ABSTRACT

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Delta cells also harboring end3Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Delta. Further comparison of ric1Delta and ypt6Delta cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Delta and ypt6Delta cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.


Subject(s)
Fungal Proteins/metabolism , Monomeric GTP-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins , Transcription Factors/metabolism , Vesicular Transport Proteins , trans-Golgi Network/chemistry , Fungal Proteins/genetics , Fungal Proteins/pharmacology , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/pharmacology , Guanine Nucleotide Exchange Factors , Intracellular Membranes/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/pharmacology , Phenotype , Protein Transport/drug effects , Qb-SNARE Proteins , SNARE Proteins , Saccharomyces cerevisiae/chemistry , Suppression, Genetic/drug effects , Transcription Factors/genetics , Transformation, Genetic , trans-Golgi Network/genetics , trans-Golgi Network/metabolism
3.
Mol Cell Biol ; 21(4): 1272-84, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11158313

ABSTRACT

The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Delta mutants for growth at 37 degrees C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans.


Subject(s)
Candida albicans/genetics , Carrier Proteins/metabolism , Cell Adhesion Molecules/genetics , Fungal Proteins/metabolism , Genes, Fungal , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Actins/metabolism , Base Sequence , Candida albicans/growth & development , Candida albicans/metabolism , Candida albicans/pathogenicity , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Cycle Proteins , Cytoskeletal Proteins , DNA Primers/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Genetic Complementation Test , Mutation , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Saccharomyces cerevisiae/growth & development , Virulence/genetics
4.
Genetics ; 154(1): 83-97, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10628971

ABSTRACT

Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.


Subject(s)
Carrier Proteins/physiology , Clathrin/metabolism , GTP-Binding Proteins , Genes, Synthetic , Golgi Apparatus/metabolism , Phosphoric Monoester Hydrolases/physiology , Saccharomyces cerevisiae/metabolism , Base Sequence , DNA Primers , Endocytosis , Inositol Polyphosphate 5-Phosphatases , Mutagenesis , Saccharomyces cerevisiae/genetics , Temperature , Vesicular Transport Proteins
5.
Arch Phys Med Rehabil ; 77(5): 512-4, 1996 May.
Article in English | MEDLINE | ID: mdl-8629931

ABSTRACT

Duchenne muscular dystrophy (DMD) is the most common neuromuscular disorder of childhood. Its clinical characteristics that derive from skeletal muscle involvement have been well described. Less well known is that visceral smooth muscle is affected in DMD. We report a case of a 19-year-old man with DMD who presented with severe nonradiating epigastric pain. He was initially sent home from the emergency department with a diagnosis of costochondritis. Acute gastric dilation was not considered in the differential diagnosis despite supportive history, physical examination findings, and radiographs. The case illustrates the lack of familiarity by clinicians of the gastrointestinal manifestations of DMD, including gastric dilatation and intestinal pseudoobstruction. Following a case discussion, the literature relevant to acute gastric atony is reviewed.


Subject(s)
Gastric Dilatation/diagnosis , Muscular Dystrophies/complications , Acute Disease , Adult , Diagnostic Errors , Gastric Dilatation/etiology , Gastric Emptying , Humans , Male
6.
Biochim Biophys Acta ; 1292(2): 249-58, 1996 Feb 08.
Article in English | MEDLINE | ID: mdl-8597570

ABSTRACT

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40-43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent Km values have been determined to be 15 microM for ATP, 1.2 microM for S6 and 10 microM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n - 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.


Subject(s)
Erythrocyte Membrane/enzymology , Protein Kinases/blood , Protein Serine-Threonine Kinases/blood , Reticulocytes/enzymology , Amino Acid Sequence , Animals , Chromatography, DEAE-Cellulose , Chromatography, Gel , Kinetics , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Octoxynol , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Mapping , Peptides/chemistry , Peptides/metabolism , Phosphopeptides/chemistry , Phosphopeptides/isolation & purification , Protein Kinases/chemistry , Protein Kinases/isolation & purification , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/isolation & purification , Rabbits , Ribosomal Protein S6 Kinases , Substrate Specificity
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