scholarly journals The cytoplasmic tail domain of the vacuolar protein sorting receptor Vps10p and a subset of VPS gene products regulate receptor stability, function, and localization.

1995 ◽  
Vol 6 (9) ◽  
pp. 1089-1102 ◽  
Author(s):  
J L Cereghino ◽  
E G Marcusson ◽  
S D Emr
Keyword(s):  
Cytoplasmic Tail ◽  
Structural Features ◽  
Receptor Protein ◽  
Carboxypeptidase Y ◽  
Type I ◽  
Mutant Form ◽  
Gene Products ◽  
Tail Domain ◽  
Vacuolar Protein ◽  
Class E

VPS10 of Saccharomyces cerevisiae encodes a type I transmembrane receptor protein required for the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY). To characterize the essential structural features and intercompartmental transport itinerary of the CPY receptor, we have constructed mutant forms of Vps10p that alter the carboxyterminal cytoplasmic tail of the protein. In addition, we have analyzed the effect these mutations as well as mutations in several VPS genes have on the function, stability, and localization of Vps10p. Although wild-type Vps10p is very stable over a 3-h chase period, overproduction of Vps10p results in PEP4-dependent degradation of the receptor. Immunofluorescence studies indicate that overexpressed receptor is delivered to the vacuole. A mutant form of Vps10p, in which 157 residues of the 164-residue cytoplasmic tail domain have been deleted, missorts CPY and is degraded rapidly. Additional mutations in the carboxy-terminus of Vps10p, including a deletion of a putative retention/recycling signal (FYVF), also result in CPY missorting and PEP4-dependent receptor instability. Because the cytoplasmic tail domain may interact with other factors, possibly VPS gene products, Vps10p stability was examined in a number of vps mutants. As was observed with the late Golgi protein Kex2p, Vps10p is unstable in a vps1 mutant. However, instability of Vps10p is even more severe in the class E vps mutants. Double mutant analyses demonstrate that this rapid degradation is dependent upon vacuolar proteases and a functional vacuolar ATPase. Fractionation studies of Vps10p in class E vps mutant strains indicate that the turnover of Vps10p occurs in a compartment other than the vacuole. These data are consistent with a model in which the cytoplasmic tail of Vps10p directs cycling of the receptor between a late Golgi sorting compartment and a prevacuolar endosome-like compartment, an exaggerated form of which is present in the vps class E mutants.

scholarly journals Vacuolar protein sorting receptor in Schizosaccharomyces pombe

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1523-1532 ◽  
Author(s):  
Tomoko Iwaki ◽  
Akira Hosomi ◽  
Sanae Tokudomi ◽  
Yoko Kusunoki ◽  
Yasuko Fujita ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Protein Sorting ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Type I ◽  
Tail Domain ◽  
Vacuolar Protein

The mechanism by which soluble proteins, such as carboxypeptidase Y, reach the vacuole in Saccharomyces cerevisiae is very similar to the mechanism of lysosomal protein sorting in mammalian cells. Vps10p is a receptor for transport of soluble vacuolar proteins in S. cerevisiae. vps10 +, a gene encoding a homologue of S. cerevisiae PEP1/VPS10, has been identified and deleted from the fission yeast Schizosaccharomyces pombe. Deletion of the vps10 + gene resulted in missorting and secretion of Sch. pombe vacuolar carboxypeptidase Cpy1p, indicating that it is required for targeting Cpy1p to the vacuole. Sch. pombe Vps10p (SpVps10p) is a type I transmembrane protein and its C-terminal cytoplasmic tail domain is essential for Cpy1p transport to the vacuole. Cells expressing green fluorescent protein-tagged SpVps10p produced a punctate pattern of fluorescence, indicating that SpVps10p was largely localized in the Golgi compartment. In addition, Sch. pombe vps26 +, vps29 + and vps35 +, encoding homologues of the S. cerevisiae retromer components VPS26, VPS29 and VPS35, were identified and deleted. Fluorescence microscopy demonstrated that SpVps10p mislocalized to the vacuolar membrane in these mutants. These results indicate that the vps26 +, vps29 + and vps35 + gene products are required for retrograde transport of SpVps10p from the prevacuolar compartment back to the Golgi in Sch. pombe cells.

scholarly journals Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and in the nucleus.

1991 ◽  
Vol 115 (5) ◽  
pp. 1293-1307 ◽  
Author(s):  
B L Bader ◽  
T M Magin ◽  
M Freudenmann ◽  
S Stumpp ◽  
W W Franke
Keyword(s):  
De Novo ◽  
Light And Electron Microscopy ◽  
Type I ◽  
Gene Products ◽  
Tail Domain ◽  
Specific Domain ◽  
Transfected Cells ◽  
Beta Actin ◽  
Actin Promoter ◽  
Nucleocytoplasmic Distribution

The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed.

scholarly journals Genomic Screen for Vacuolar Protein Sorting Genes inSaccharomyces cerevisiae

2002 ◽  
Vol 13 (7) ◽  
pp. 2486-2501 ◽  
Author(s):  
Cecilia J. Bonangelino ◽  
Edna M. Chavez ◽  
Juan S. Bonifacino
Keyword(s):  
Alkaline Phosphatase ◽  
Actin Cytoskeleton ◽  
Protein Sorting ◽  
Carboxypeptidase Y ◽  
Gene Products ◽  
The Novel ◽  
Vacuolar Protein Sorting ◽  
Novel Gene ◽  
A Genome ◽  
Vacuolar Protein

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains ofSaccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature α-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30°C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.

scholarly journals Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant.

1996 ◽  
Vol 7 (6) ◽  
pp. 985-999 ◽  
Author(s):  
S E Rieder ◽  
L M Banta ◽  
K Köhrer ◽  
J M McCaffery ◽  
S D Emr
Keyword(s):  
Protein Sorting ◽  
Retrograde Transport ◽  
Carboxypeptidase Y ◽  
Coat Proteins ◽  
Electron Microscopic ◽  
Yeast Saccharomyces Cerevisiae ◽  
Intense Staining ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein ◽  
Class E

In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.

scholarly journals Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases.

1996 ◽  
Vol 133 (3) ◽  
pp. 529-541 ◽  
Author(s):  
A A Cooper ◽  
T H Stevens
Keyword(s):  
Amino Acid ◽  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Carboxypeptidase Y ◽  
Type I ◽  
Binding Studies ◽  
Cytosolic Domain ◽  
Sorting Receptor ◽  
Vacuolar Protein

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.

scholarly journals Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

1997 ◽  
Vol 137 (1) ◽  
pp. 79-92 ◽  
Author(s):  
Matthew N.J. Seaman ◽  
Eric G. Marcusson ◽  
Joan Lin Cereghino ◽  
Scott D. Emr
Keyword(s):  
Transmembrane Protein ◽  
Protein Sorting ◽  
Vacuolar Membrane ◽  
Carboxypeptidase Y ◽  
Gene Products ◽  
C Elegans ◽  
Conditional Allele ◽  
Proteinase A ◽  
Sorting Receptor ◽  
Vacuolar Protein

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.

scholarly journals VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae.

1995 ◽  
Vol 131 (3) ◽  
pp. 603-617 ◽  
Author(s):  
R C Piper ◽  
A A Cooper ◽  
H Yang ◽  
T H Stevens
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Golgi Compartment ◽  
Prevacuolar Compartment ◽  
Sensitive Allele ◽  
Vacuolar Protein ◽  
Endocytic Traffic ◽  
Class E

Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps. Endocytosed proteins are also delivered to the vacuole. It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole. One group of genes has been described that appears to control both of these pathways. Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment. We have characterized one of the class E VPS genes, VPS27, in detail to address this question. Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment. Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY. Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate. Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells.

Comparative surface and ultrastructural views of methylotrophic bacteria by TEM, STEM, SE, and freeze-etch electron microscopy.

1987 ◽  
Vol 45 ◽  
pp. 792-793
Author(s):  
T.A. Fassel ◽  
M.J. Schaller ◽  
M.E. Lidstrom ◽  
C.C. Remsen
Keyword(s):  
Cytoplasmic Membrane ◽  
Structural Features ◽  
Methylotrophic Bacteria ◽  
Type I ◽  
Type Ii ◽  
Membrane Complex ◽  
Oxidation Of Methane ◽  
Methane Oxidizing Bacteria ◽  
Wall Structures ◽  
Methylomonas Albus

Methylotrophic bacteria play an Important role in the environment in the oxidation of methane and methanol. Extensive intracytoplasmic membranes (ICM) have been associated with the oxidation processes in methylotrophs and chemolithotrophic bacteria. Classification on the basis of ICM arrangement distinguishes 2 types of methylotrophs. Bundles or vesicular stacks of ICM located away from the cytoplasmic membrane and extending into the cytoplasm are present in Type I methylotrophs. In Type II methylotrophs, the ICM form pairs of peripheral membranes located parallel to the cytoplasmic membrane. Complex cell wall structures of tightly packed cup-shaped subunits have been described in strains of marine and freshwater phototrophic sulfur bacteria and several strains of methane oxidizing bacteria. We examined the ultrastructure of the methylotrophs with particular view of the ICM and surface structural features, between representatives of the Type I Methylomonas albus (BG8), and Type II Methylosinus trichosporium (OB-36).

scholarly journals Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

Virology ◽  
2016 ◽  
Vol 490 ◽  
pp. 1-5 ◽  
Author(s):  
Nora F. Mahmoud ◽  
Chyntia Jasirwan ◽  
Satoshi Kanemoto ◽  
Aika Wakata ◽  
Bochao Wang ◽  
...  
Keyword(s):  
Cytoplasmic Tail ◽  
Glycoprotein B ◽  
Tail Domain
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