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1.
Sci Rep ; 11(1): 8007, 2021 04 13.
Article in English | MEDLINE | ID: mdl-33850201

ABSTRACT

The AP1/σ1B knockout causes impaired synaptic vesicle recycling and enhanced protein sorting into endosomes, leading to severe intellectual disability. These disturbances in synaptic protein sorting induce as a secondary phenotype the upregulation of AP2 CCV mediated endocytosis. Synapses contain canonical AP2 CCV and AP2 CCV with a more stable coat and thus extended life time. In AP1/σ1B knockout synapses, pool sizes of both CCV classes are doubled. Additionally, stable CCV of the knockout are more stabilised than stable wt CCV. One mechanism responsible for enhanced CCV stabilisation is the reduction of synaptojanin1 CCV levels, the PI-4,5-P2 phosphatase essential for AP2 membrane dissociation. To identify mechanisms regulating synaptojanin1 recruitment, we compared synaptojanin1 CCV protein interactome levels and CCV protein interactions between both CCV classes from wt and knockout mice. We show that ITSN1 determines synaptojanin1 CCV levels. Sgip1/AP2 excess hinders synaptojanin1 binding to ITSN1, further lowering its levels. ITSN1 levels are determined by Eps15, not Eps15L1. In addition, the data reveal that reduced amounts of pacsin1 can be counter balanced by its enhanced activation. These data exemplify the complexity of CCV life cycle regulation and indicate how cargo proteins determine the life cycle of their CCV.


Subject(s)
Adaptor Proteins, Signal Transducing , Nerve Tissue Proteins , Phosphoric Monoester Hydrolases , Synaptic Vesicles , Animals , Endosomes , Mice
2.
J Cell Biol ; 152(3): 595-606, 2001 Feb 05.
Article in English | MEDLINE | ID: mdl-11157985

ABSTRACT

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.


Subject(s)
Adaptor Protein Complex 1 , Adaptor Protein Complex 2 , Adaptor Protein Complex mu Subunits , Cell Polarity , Clathrin/metabolism , Epithelial Cells/physiology , Membrane Proteins/metabolism , Protein Transport/physiology , Transport Vesicles/metabolism , Adaptor Protein Complex alpha Subunits , Adaptor Protein Complex gamma Subunits , Adaptor Proteins, Vesicular Transport , Adenoviridae/genetics , Adenoviridae/metabolism , Animals , Epithelial Cells/ultrastructure , Furin , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Membrane Proteins/genetics , Protein Subunits , Receptors, LDL/metabolism , Subtilisins/metabolism , Swine , Transfection , Transport Vesicles/chemistry , Transport Vesicles/ultrastructure
3.
J Biol Chem ; 276(15): 11606-14, 2001 Apr 13.
Article in English | MEDLINE | ID: mdl-11152450

ABSTRACT

Vacuolar aminopeptidase 1 is transported to the vacuole by cytoplasmic double-membrane vesicles, the nonclassic Cvt pathway. The cytosolic protein dodecamerizes and is enclosed in a double-membrane vesicle, which is transported to and fuses with the vacuole releasing a single-membrane autophagic body into the vacuolar lumen. This is degraded and the precursor sequence of aminopeptidase 1 is removed. This pathway resembles autophagy, and most proteins identified to function in the Cvt pathway are also required for autophagy and vice versa. The cytosolic precursor protein and the matured vacuolar protein form a homododecameric complex, and only this complex has enzymatic activity. We developed a new genetic screen to isolate mutants in the biogenesis of vacuolar aminopeptidase 1 based on its enzymatic activity. The sensitivity of this assay made it possible for us to search for mutants under conditions where autophagy is down-regulated, and we describe two new mutants defective in the biogenesis pathway of vacuolar aminopeptidase 1. Mutants are defective in dodecamerization of pApe1p and in Cvt vesicle formation. Complex assembly and transport vesicle formation appear to be linked processes. This mechanism can control the potentially harmful cytoplasmic proteolytic activity and could be the driving force for this nonclassic mechanism of vacuolar enzyme transport.


Subject(s)
Aminopeptidases/metabolism , Mutation , Vacuoles/enzymology , Aminopeptidases/chemistry , Aminopeptidases/genetics , Biological Transport , Biopolymers , Protein Conformation
4.
Pharmacogenomics J ; 1(4): 262-71, 2001.
Article in English | MEDLINE | ID: mdl-11908769

ABSTRACT

Protein transport and sorting in the secretory and endocytic pathways via vesicles is required for organelle biogenesis, constitutive and regulated secretion and constitutive and regulated endocytosis. It is essential for a multicellular organism and the function of its specialised cell types that the multiple transport and sorting events are highly accurate. They determine the protein and lipid composition of specialised compartments, receptor protein function and membrane homeostasis. This review describes the individual events involved in the process of vesicle mediated protein transport and sorting and summarizes the knowledge about the function of proteins and lipids orchestrating the process.


Subject(s)
Protein Transport/physiology , Transport Vesicles/physiology , Capsid/metabolism , Capsid/pharmacokinetics , Clathrin/metabolism , Clathrin/pharmacokinetics , Lipids/physiology , Transport Vesicles/metabolism
5.
J Cell Sci ; 114(Pt 24): 4469-76, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11792812

ABSTRACT

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in mu1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In mu1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.


Subject(s)
Adaptor Protein Complex 1 , Adaptor Protein Complex mu Subunits , Carrier Proteins/genetics , Clathrin/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Receptor, IGF Type 2/metabolism , Up-Regulation/genetics , Adaptor Proteins, Vesicular Transport , Animals , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Endocytosis/genetics , Endosomes/genetics , Endosomes/metabolism , Exocytosis/genetics , Mice , Mice, Knockout , Protein Transport/genetics
6.
EMBO J ; 19(10): 2193-203, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10811610

ABSTRACT

The heterotetrameric AP-1 complex is involved in the formation of clathrin-coated vesicles at the trans-Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse mu1A-adaptin gene causes embryonic lethality at day 13.5. In cells deficient in micro1A-adaptin the remaining AP-1 adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of micro1A-adaptin-deficient embryos that show gamma-adaptin binding to membranes, indicating the formation of an epithelial specific AP-1B complex and demonstrating the absence of additional mu1A homologs. Mannose 6-phosphate receptors are cargo molecules that exit the TGN via AP-1-clathrin-coated vesicles. The steady-state distribution of the mannose 6-phosphate receptors MPR46 and MPR300 in mu1A-deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP-1 is required for retrograde endosome to TGN transport of the receptor.


Subject(s)
Adaptor Protein Complex 1 , Adaptor Protein Complex mu Subunits , Clathrin/metabolism , Membrane Proteins/deficiency , Receptor, IGF Type 2/metabolism , Adaptor Protein Complex alpha Subunits , Adaptor Proteins, Vesicular Transport , Animals , Biological Transport , Clathrin/genetics , Embryonic and Fetal Development/genetics , Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Mice , Receptor, IGF Type 2/genetics
7.
FEBS Lett ; 470(3): 232-8, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10745074

ABSTRACT

Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane.


Subject(s)
Aminopeptidases/metabolism , Cytoplasm/metabolism , Fungal Proteins/physiology , HSP70 Heat-Shock Proteins/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Adenosine Triphosphatases , Aminopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Biological Transport , Enzyme Precursors/chemistry , Enzyme Precursors/metabolism , Fluorescent Antibody Technique , Fungal Proteins/genetics , Gene Deletion , Genes, Fungal/genetics , Genes, Fungal/physiology , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Response , Intracellular Membranes/metabolism , Molecular Weight , Phagocytosis , Protein Binding , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology
8.
Comp Biochem Physiol B Biochem Mol Biol ; 127(4): 433-41, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11281260

ABSTRACT

The existence of two homologous mannose 6-phosphate receptors (MPRs) with overlapping, but distinct functions has raised the question of at what stage in the phylogenetic tree the two receptors have occurred for the first time. In this paper, we present a partial cDNA sequence of Mr 300 kDa MPR (MPR 300) from poeciliid fish (Xiphophorus). It contains a 5'-untranslated region followed by the initiator ATG, and an open reading frame that corresponds to cassettes 1-5 and part of cassette 6 of mammalian MPR 300. The size of the mRNA transcript for fish MPR 300 was comparable with that of other vertebrates. The amino acid sequence of fish MPR 300 displays 48-52% similarity with mammalian and chicken MPR 300. In particular, all the cysteine residues involved in disulfide bonding and an arginine residue, which is considered to be part of the mannose 6-phosphate binding site in cassette 3 of mammalian MPR 300, are conserved. Sequence similarities were significantly higher within cassette 3 and within cassette 5, to which a ligand-binding function has not yet been ascribed. Sequence similarities of the internal cassettes of MPR 300 are discussed with regard to the multifunctional nature of MPR 300.


Subject(s)
Conserved Sequence/genetics , DNA, Complementary/analysis , Poecilia/genetics , Receptor, IGF Type 2/genetics , Amino Acid Sequence , Animals , Blotting, Northern , Cattle , Chickens , DNA Primers/chemistry , Humans , Mice , Molecular Sequence Data , Molecular Weight , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid
9.
Mol Biol Cell ; 10(9): 2879-89, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10473633

ABSTRACT

In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins. The physiological condition of the cell often determines the degradation pathway of a specific protein. In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles. After starvation of the cells, protein uptake increases. Uptake and degradation are temperature dependent and show biphasic kinetics. Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles. Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase. We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway. This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.


Subject(s)
Cytosol/metabolism , Fungal Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Biological Transport , Biomarkers , Cytosol/ultrastructure , Endopeptidases/metabolism , Fungal Proteins/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Response , Isoenzymes/metabolism , Kinetics , Microscopy, Electron , Proton-Translocating ATPases/antagonists & inhibitors , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/ultrastructure , Temperature , Vacuoles/chemistry , Vacuoles/enzymology , Vacuoles/ultrastructure
10.
J Biol Chem ; 274(9): 5385-90, 1999 Feb 26.
Article in English | MEDLINE | ID: mdl-10026148

ABSTRACT

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the gamma-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. gamma-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free beta1- or micro1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.


Subject(s)
Fetal Death/genetics , Genes, Lethal , Membrane Proteins/genetics , Adaptor Protein Complex gamma Subunits , Animals , Heterozygote , Mice , Mutagenesis, Site-Directed , Phenotype
11.
Proc Natl Acad Sci U S A ; 95(23): 13453-8, 1998 Nov 10.
Article in English | MEDLINE | ID: mdl-9811821

ABSTRACT

Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruffled borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts in vitro revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.


Subject(s)
Bone Resorption/metabolism , Cathepsins/deficiency , Cathepsins/genetics , Osteopetrosis/metabolism , Animals , Cathepsin K , Disease Models, Animal , Mice , Mice, Knockout , Osteoclasts/metabolism , Osteopetrosis/genetics
12.
Mol Cell Biol ; 16(5): 2369-77, 1996 May.
Article in English | MEDLINE | ID: mdl-8628304

ABSTRACT

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.


Subject(s)
Carrier Proteins/metabolism , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/physiology , Vacuoles/metabolism , Vesicular Transport Proteins , Alleles , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Cloning, Molecular , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Genes, Fungal , Genetic Complementation Test , Guanine Nucleotide Exchange Factors , Humans , Mammals , Molecular Sequence Data , Mutagenesis , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/ultrastructure , Sequence Homology, Amino Acid , Temperature , Vacuoles/ultrastructure
13.
Cryobiology ; 32(4): 379-88, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7656571

ABSTRACT

In this article we describe the intensive investigation of defined physicochemical parameters for their influence on the preparation, storage stability in liquid nitrogen, and the post-thaw productivity of a recombinant strain of Saccharomyces cerevisiae expressing the human factor XIIIa protein. Preparation of industrially sized seed cultures and their storage stability were monitored over more than a 1-year period. Major parameters recorded before and after thawing were number of colony-forming units on agar slant media, plasmid retention, and expression capacity of the recombinant protein. The viability of the cells after reconstitution of the frozen batches was found to improve significantly if a certain combination of prethaw growth conditions was applied and amino acids were included in the cryoprotectant media. In addition, the influence of different concentrations of glycerol as a cryoprotectant was investigated and the specific freezing conditions which improved the viability were identified. Even without complex freezing devices for the controlled freezing of cultures, viability rates of 85% and higher were obtained, enabling consistent production of the recombinant protein.


Subject(s)
Cryopreservation/methods , Saccharomyces cerevisiae , Amino Acids , Culture Media/chemistry , Gene Expression , Glucose , Humans , Mutation , Plasmids , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombination, Genetic , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Salts , Time Factors , Transglutaminases/biosynthesis , Transglutaminases/genetics
14.
J Antibiot (Tokyo) ; 47(11): 1188-94, 1994 Nov.
Article in English | MEDLINE | ID: mdl-8002380

ABSTRACT

A new salicylic acid derivative, caloporoside, was isolated from fermentations of Caloporus dichrous. Its structure was elucidated by a combination of chemical and spectroscopic methods. Caloporoside exhibits weak antibacterial and antifungal activities and is a quite selective inhibitor of phospholipase C isolated from pig brain (Ki 12, 3 microM).


Subject(s)
Basidiomycota/metabolism , Mannose/analogs & derivatives , Salicylates/isolation & purification , Type C Phospholipases/antagonists & inhibitors , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Mannose/chemistry , Mannose/isolation & purification , Mannose/pharmacology , Salicylates/chemistry , Salicylates/pharmacology , Swine
15.
EMBO J ; 12(5): 2195-204, 1993 May.
Article in English | MEDLINE | ID: mdl-8387919

ABSTRACT

The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) are required for the sorting of soluble hydrolases to the yeast vacuole. Over-production of Vps34p suppresses the growth and vacuolar protein sorting defects associated with vps15 kinase domain mutants, suggesting that Vps15p and Vps34p functionally interact. Subcellular fractionation and sucrose density gradients indicate that Vps15p is responsible for the association of Vps34p with an intracellular membrane fraction. Chemical cross-linking and native immunoprecipitation experiments demonstrate that Vps15p and Vps34p interact as components of a hetero-oligomeric protein complex. In addition, we show that an intact Vps15 protein kinase domain is required for activation of the Vps34 PI 3-kinase, suggesting that the Vps34 lipid kinase is regulated by a Vps15p-mediated protein phosphorylation event. We propose that Vps15p and Vps34p function together as components of a membrane-associated signal transduction complex that regulates intracellular protein trafficking decisions through protein and lipid phosphorylation events.


Subject(s)
Fungal Proteins/metabolism , Phosphotransferases/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/enzymology , Vacuoles/enzymology , Biological Transport , Cell Fractionation , Cell Membrane/enzymology , Enzyme Activation , Lysosomes/enzymology , Multienzyme Complexes/metabolism , Mutation , Myristic Acid , Myristic Acids/metabolism , Phosphatidylinositol 3-Kinases , Phosphotransferases/biosynthesis , Protein Serine-Threonine Kinases/genetics
16.
Science ; 260(5104): 88-91, 1993 Apr 02.
Article in English | MEDLINE | ID: mdl-8385367

ABSTRACT

The VPS34 gene product (Vps34p) is required for protein sorting to the lysosome-like vacuole of the yeast Saccharomyces cerevisiae. Vps34p shares significant sequence similarity with the catalytic subunit of bovine phosphatidylinositol (PI) 3-kinase [the 110-kilodalton (p110) subunit of PI 3-kinase], which is known to interact with activated cell surface receptor tyrosine kinases. Yeast strains deleted for the VPS34 gene or carrying vps34 point mutations lacked detectable PI 3-kinase activity and exhibited severe defects in vacuolar protein sorting. Overexpression of Vps34p resulted in an increase in PI 3-kinase activity, and this activity was specifically precipitated with antisera to Vps34p. VPS34 encodes a yeast PI 3-kinase, and this enzyme appears to regulate intracellular protein trafficking decisions.


Subject(s)
Fungal Proteins/metabolism , Genes, Fungal , Phosphotransferases/genetics , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Animals , Brain/enzymology , Cattle , Chromatography, High Pressure Liquid , Gene Deletion , Gene Expression , Lysosomes/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphatidylinositol 3-Kinases , Phosphotransferases/chemistry , Phosphotransferases/metabolism , Point Mutation , Saccharomyces cerevisiae/enzymology , Sequence Homology, Amino Acid , Signal Transduction , Vacuoles/metabolism
17.
FEBS Lett ; 283(1): 78-84, 1991 May 20.
Article in English | MEDLINE | ID: mdl-2037077

ABSTRACT

The gene of the proteinase yscA inhibitor IA3, PAI3, of the yeast Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library and sequenced. The gene codes for a single protein of 68 amino acids. The structural PAI3 gene was deleted in vitro by oligonucleotide-site-directed mutagenesis. The mutated allele was introduced via homologous recombination into the genome of wild-type yeast and into the genome of a yeast mutant, which lacks the second cytoplasmic proteinase-inhibitor, IB2. The deficiency of either or of both inhibitors has no effect on the cell viability under various physiological conditions. The inhibitor mutants, however, show an increase in the general in vivo protein degradation rate. The IA3 mutant has a 2-3-fold increased protein degradation rate in the first 6 h after a shift from rich medium onto starvation-medium, whereas the IB2 mutant shows a constantly increased degradation rate of 20-50% under the same conditions. The inhibitor double null mutant has the same protein degradation rate as the IA3 null mutant. These results suggest an in vivo interaction between the vacuolor endopeptidases and their cytoplasmic inhibitors.


Subject(s)
Protease Inhibitors , Saccharomyces cerevisiae/genetics , Serine Proteinase Inhibitors , Amino Acid Sequence , Base Sequence , DNA, Fungal/genetics , Genes, Fungal , Molecular Sequence Data , Mutation , Nucleic Acid Hybridization , Saccharomyces cerevisiae/physiology , Serine Endopeptidases/genetics
18.
Eur J Biochem ; 197(1): 1-7, 1991 Apr 10.
Article in English | MEDLINE | ID: mdl-2015812

ABSTRACT

The gene for proteinase yscB inhibitor I2B (PBI2) from Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library, and was sequenced. The gene codes for a single protein of 75 amino acids. In contrast to the published amino acid sequence [Maier, K., Müller, H., Tesch, R., Trolp, T., Witt, I. & Holzer, H. (1979) J. Biol. Chem. 254, 12,555-12,561] the DNA sequence revealed a valine instead of a leucine at position 33 (32 of the mature protein). Therefore the primary sequences of the isoinhibitors I2B of S. cerevisiae and I1B of Saccharomyces carlsbergensis differ only at position 34 (glutamic acid/lysine). The open reading frame of PBI2 was replaced in vitro by the URA3 gene and a I2B null mutant of S. cerevisiae was constructed by gene replacement. The mutation resulted in an elevation of the protein degradation rate by 50% when grown under nutritional stress compared to the isogenic wild type. Growth and viability of the cells was not significantly affected by the absence of I2B.


Subject(s)
Genes, Fungal , Repressor Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Serine Endopeptidases , Serine Proteinase Inhibitors , Serine Proteinase Inhibitors/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Fungal/genetics , DNA, Fungal/isolation & purification , Escherichia coli/genetics , Gene Library , Genotype , Kinetics , Molecular Sequence Data , Restriction Mapping , Serine Proteinase Inhibitors/pharmacology , Vesicular Transport Proteins
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