scholarly journals A Multispecificity Syntaxin Homologue, Vam3p, Essential for Autophagic and Biosynthetic Protein Transport to the Vacuole

1997 ◽  
Vol 138 (3) ◽  
pp. 517-529 ◽  
Author(s):  
Tamara Darsow ◽  
Stephanie E. Rieder ◽  
Scott D. Emr
Keyword(s):  
Protein Transport ◽  
Temperature Shift ◽  
Subcellular Fractionation ◽  
Polyclonal Antiserum ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Fusion Reactions ◽  
Vesicle Docking ◽  
Transport Vesicle ◽  
Mutant Phenotypes

Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283–amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3tsf). Upon shifting the vam3tsf mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3tsf cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3tsf cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Δ mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Δ mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

scholarly journals Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment.

1993 ◽  
Vol 121 (6) ◽  
pp. 1245-1256 ◽  
Author(s):  
T A Vida ◽  
G Huyer ◽  
S D Emr
Keyword(s):  
Golgi Complex ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Subcellular Fractionation ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Potential Donor ◽  
Transport Vesicle

We are studying intercompartmental protein transport to the yeast lysosome-like vacuole with a reconstitution assay using permeabilized spheroplasts that measures, in an ATP and cytosol dependent reaction, vacuolar delivery and proteolytic maturation of the Golgi-modified precursor forms of vacuolar hydrolases like carboxypeptidase Y (CPY). To identify the potential donor compartment in this assay, we used subcellular fractionation procedures that have uncovered a novel membrane-enclosed prevacuolar transport intermediate. Differential centrifugation was used to separate permeabilized spheroplasts into 15K and 150K g membrane pellets. Centrifugation of these pellets to equilibrium on sucrose density gradients separated vacuolar and Golgi complex marker enzymes into light and dense fractions, respectively. When the Golgi-modified precursor form of CPY (p2CPY) was examined (after a 5-min pulse, 30-s chase), as much as 30-40% fractionated with an intermediate density between both the vacuole and the Golgi complex. Pulse-chase labeling and fractionation of membranes indicated that p2CPY in this gradient region had already passed through the Golgi complex, which kinetically ordered it between the Golgi and the vacuole. A mutant CPY protein that lacks a functional vacuolar sorting signal was detected in Golgi fractions but not in the intermediate compartment indicating that this corresponds to a post-sorting compartment. Based on the low transport efficiency of the mutant CPY protein in vitro (decreased by sevenfold), this intermediate organelle most likely represents the donor compartment in our reconstitution assay. This organelle is not likely to be a transport vesicle intermediate because EM analysis indicates enrichment of 250-400 nm compartments and internalization of surface-bound 35S-alpha-factor at 15 degrees C resulted in its apparent cofractionation with wild-type p2CPY, indicating an endosome-like compartment (Singer, B., and H. Reizman. 1990. J. Cell Biol. 110:1911-1922). Fractionation of p2CPY accumulated in the temperature sensitive vps15 mutant revealed that the vps15 transport block did not occur in the endosome-like compartment but rather in the late Golgi complex, presumably the site of CPY sorting. Therefore, as seen in mammalian cells, yeast CPY is sorted away from secretory proteins in the late Golgi and transits to the vacuole via a distinct endosome-like intermediate.

Genetic Interactions Between a pep7 Mutation and the PEP12 and VPS45 Genes: Evidence for a Novel SNARE Component in Transport Between the Saccharomyces cerevisiae Golgi Complex and Endosome

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 467-478 ◽  
Author(s):  
Gene C Webb ◽  
Marloes Hoedt ◽  
Lynn J Poole ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Complex ◽  
Genetic Interactions ◽  
Snare Complex ◽  
Carboxypeptidase Y ◽  
Fusion Reactions ◽  
Vesicle Docking ◽  
Transport Vesicles ◽  
Allele Specific ◽  
Mutant Phenotypes

The PEP7 gene from Saccharomyces cerevisiae encodes a 59-kD hydrophilic polypeptide that is required for transport of soluble vacuolar hydrolase precursors from the TGN to the endosome. This study presents the results of a high-copy suppression analysis of pep7-20 mutant phenotypes. This analysis demonstrated that both VPS45 and PEP12 are allele-specific high-copy suppressors of pep7-20 mutant phenotypes. Overexpression of VPS45 was able to completely suppress the Zn2+ sensitivity and partially suppress the carboxypeptidase Y deficiency. Overexpression of PEP12 was able to do the same, but to a lesser extent. Vps45p and Pep12p are Sec1p and syntaxin (t-SNARE) homologues, respectively, and are also thought to function in transport between the TGN and endosome. Two additional vacuole pathway SNARE complex homologues, Vps33p (Sec1p) and Pth1p (syntaxin), when overexpressed, were unable to suppress pep7-20 or any other pep7 allele, further supporting the specificity of the interactions of pep7-20 with PEP12 and VPS45. Because several other vesicle docking/fusion reactions take place in the cell without discernible participation of Pep7p homologues, we suggest that Pep7p is a step-specific regulator of docking and/or fusion of TGN-derived transport vesicles onto the endosome.

scholarly journals Effects of a temperature-sensitiveMinutemutation on gene expression inDrosophila melanogaster

1984 ◽  
Vol 43 (3) ◽  
pp. 257-275 ◽  
Author(s):  
Donald A. R. Sinclair ◽  
Thomas A. Grigliatti ◽  
Thomas C. Kaufman
Keyword(s):  
Gene Expression ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Products ◽  
The Past ◽  
Reciprocal Temperature ◽  
Sex Comb ◽  
Mutant Phenotypes

SUMMARYMinute(M) lesions exhibit a striking propensity for interacting with many different mutations. In the past, few attempts have been made to explain these diverse phenomena. This study describes a variety of temperature-sensitive (ts) interactions exhibited by the ts third chromosomeMinutemutationM(3)LS4Q-III(Q-III). Most of these interactions (i.e. those involvingvg, cp, Dl, DfdorLy) reflectQ-III-induced enhancement of the respective mutant phenotypes at the restrictive temperature. However,Q-IIIalso suppresses the extra-sex-comb phenotypes ofPcandMscat 29 °C and evokes lethal and bristle traits when combined withJ34eat the restrictive temperature. All of these interactions are characteristic of non-tsMinutelesions and thus they appear to be correlated with general physiological perturbations associated with theMsyndrome. In addition, our findings show that mutations that affect ribosome production and/or function, namelysu(f)ts67gandbbts−1, exhibit interactions comparable to those elicited byQ-III. Hence, in accordance with previous findings, we argue that most of theQ-IIIinteractions can be attributed to reduced translational capacity at the restrictive temperature. Finally, reciprocal temperature shift studies were used to delineate TSPs for interactions betweenQ-IIIandvg(mid to late second instar),cp(about mid-third instar),Dfd(early third instar) andDl(late second to mid third instar). We believe that these TSPs represent developmental intervals during which the respective gene products are utilized.

scholarly journals Novel PtdIns(3)P-binding protein Etf1 functions as an effector of the Vps34 PtdIns 3-kinase in autophagy

2002 ◽  
Vol 158 (4) ◽  
pp. 761-772 ◽  
Author(s):  
Andrew E. Wurmser ◽  
Scott D. Emr
Keyword(s):  
Protein Trafficking ◽  
Transmembrane Protein ◽  
Genetic Selection ◽  
Basic Amino Acid ◽  
Current Evidence ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Phosphatidylinositol 3 Kinase ◽  
Transport Vesicle ◽  
Autophagy Pathway

Autophagy is the process whereby cytoplasmic cargo (e.g., protein and organelles) are sequestered within a double membrane–enclosed transport vesicle and degraded after vesicle fusion with the vacuole/lysosome. Current evidence suggests that the Vps34 phosphatidylinositol 3-kinase is essential for macroautophagy, a starvation-induced autophagy pathway (Kihara et al., 2001). Here, we characterize a requirement for Vps34 in constitutive autophagy by the cytoplasm-to-vacuole targeting (Cvt) pathway. First, we show that transient disruption of phosphatidylinositol (PtdIns) 3-phosphate (PtdIns[3]P) synthesis through inactivation of temperature-sensitive Vps34 or its upstream activator, Vps15, blocks the Cvt and macroautophagy pathways. Yet, PtdIns(3)P-binding FYVE domain-containing proteins, which mediate carboxypeptidase Y (CPY) transport to the vacuole by the CPY pathway, do not account for the requirement of Vps34 in autophagy. Using a genetic selection designed to isolate PtdIns(3)P-binding effectors of Vps34, we identify Etf1, an uncharacterized type II transmembrane protein. Although Etf1 does not contain a known 3-phosphoinositide–binding domain (i.e., FYVE or Phox), we find that Etf1 interacts with PtdIns(3)P and that this interaction requires a basic amino acid motif (KKPAKK) within the cytosolic region of the protein. Moreover, deletion of ETF1 or mutation of the KKPAKK motif results in strong sorting defects in the Cvt pathway but not in macroautophagy or in CPY sorting. We propose that Vps34 regulates the CPY, Cvt, and macroautophagy pathways through distinct sets of PtdIns(3)P-binding effectors and that Vps34 promotes protein trafficking in the Cvt pathway through activation/localization of the effector protein Etf1.

scholarly journals Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant.

1991 ◽  
Vol 114 (2) ◽  
pp. 207-218 ◽  
Author(s):  
T R Graham ◽  
S D Emr
Keyword(s):  
Golgi Complex ◽  
Protein Modification ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Temperature Shift ◽  
Specific Protein ◽  
Carboxypeptidase Y ◽  
Protein Functions ◽  
Dependent Transport ◽  
Vacuolar Protein

The sec18 and sec23 secretory mutants of Saccharomyces cerevisiae have previously been shown to exhibit temperature-conditional defects in protein transport from the ER to the Golgi complex (Novick, P., S. Ferro, and R. Schekman, 1981. Cell. 25:461-469). We have found that the Sec18 and Sec23 protein functions are rapidly inactivated upon shifting mutant cells to the nonpermissive temperature (less than 1 min). This has permitted an analysis of the potential role these SEC gene products play in transport events distal to the ER. The sec-dependent transport of alpha-factor (alpha f) and carboxypeptidase Y (CPY) biosynthetic intermediates present throughout the secretory pathway was monitored in temperature shift experiments. We found that Sec18p/NSF function was required sequentially for protein transport from the ER to the Golgi complex, through multiple Golgi compartments and from the Golgi complex to the cell surface. In contrast, Sec23p function was required in the Golgi complex, but only for transport of alpha f out of an early compartment. Together, these studies define at least three functionally distinct Golgi compartments in yeast. From cis to trans these compartments contain: (a) An alpha 1----6 mannosyltransferase; (b) an alpha 1----3 mannosyltransferase; and (c) the Kex2 endopeptidase. Surprisingly, we also found that a pool of Golgi-modified CPY (p2 CPY) located in a compartment distal to the alpha 1----3 mannosyltransferase does not require Sec18p function for final delivery to the vacuole. This compartment appears to be equivalent to the Kex2 compartment as we show that a novel vacuolar CPY-alpha f-invertase fusion protein undergoes efficient Kex2-dependent cleavage resulting in the secretion of invertase. We propose that this Kex2 compartment is the site in which vacuolar proteins are sorted from proteins destined to be secreted.

scholarly journals Synthetic Genetic Interactions With Temperature-Sensitive Clathrin in Saccharomyces cerevisiae: Roles for Synaptojanin-Like Inp53p and Dynamin-Related Vps1p in Clathrin-Dependent Protein Sorting at the trans-Golgi Network

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 83-97
Author(s):  
Eric S Bensen ◽  
Giancarlo Costaguta ◽  
Gregory S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Protein Sorting ◽  
Genetic Interactions ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Trans Golgi Network ◽  
Vacuolar Protein ◽  
Golgi Network

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 α-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 α-factor maturation defects and exacerbated α-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.

scholarly journals Inhibition of Endosome Function in CHO Cells Bearing a Temperature-sensitive Defect in the Coatomer (COPI) Component ε-COP

1997 ◽  
Vol 139 (7) ◽  
pp. 1747-1759 ◽  
Author(s):  
Elizabeth Daro ◽  
David Sheff ◽  
Marie Gomez ◽  
Thomas Kreis ◽  
Ira Mellman
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Temperature Shift ◽  
Endocytic Pathway ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Intact Cells ◽  
Early Endosomes ◽  
Mutant Phenotypes

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit ε-COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable ε-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type ε-COP cDNA demonstrating that they directly or indirectly reflected the ε-COP defect. Taken together, the results suggest that ε-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

scholarly journals TEMPERATURE-SENSITIVE MUTATIONS OF THE NOTCH LOCUS IN DROSOPHILA MELANOGASTER

Genetics ◽  
1975 ◽  
Vol 81 (1) ◽  
pp. 143-162 ◽  
Author(s):  
David L Shellenbarger ◽  
J Dawson Mohler
Keyword(s):  
Temperature Shift ◽  
Temperature Sensitive ◽  
Spatial Differences ◽  
Region Temperature ◽  
Complex Locus ◽  
Notch Locus ◽  
Temporal And Spatial ◽  
Chemical Conditions ◽  
Interallelic Complementation ◽  
The Right

ABSTRACT Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)Nts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax59d5 is found to be temperature-sensitive, while fag, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fag, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fag, spl, and l(1)N cause defects at specific, non-overlapping times in development.—We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.

scholarly journals Identification and Characterization of Genes That Interact With lin-12 in Caenorhabditis elegans

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1675-1695 ◽  
Author(s):  
Frans E Tax ◽  
James H Thomas ◽  
Edwin L Ferguson ◽  
H Robert Horvitzt
Keyword(s):  
Cell Fate ◽  
Low Frequency ◽  
Signal Transduction Pathway ◽  
Temperature Shift ◽  
Egg Laying ◽  
Null Alleles ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Suppressor Mutations

Abstract We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-l7, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup1 7 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup1 7and lag-2, suggest that both genes act at approximately the same time as lin-12in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

scholarly journals MATERNAL-ZYGOTIC GENE INTERACTIONS DURING FORMATION OF THE DORSOVENTRAL PATTERN IN DROSOPHILA EMBRYOS

Genetics ◽  
1983 ◽  
Vol 105 (3) ◽  
pp. 615-632 ◽  
Author(s):  
Pat Simpson
Keyword(s):  
Early Embryo ◽  
Drosophila Embryo ◽  
Sensitive Period ◽  
Temperature Sensitive ◽  
Mutant Genotype ◽  
Dominant Phenotype ◽  
Zygotic Gene ◽  
Definition Of ◽  
Mutant Phenotypes ◽  
Dominant Lethality

ABSTRACT Maternal-zygotic interactions involving the three genes dorsal (dl), twist (twi) and snail (sna) are described. The results suggest that all three are involved in the process by which the dorsoventral pattern of the Drosophila embryo is established. First, the lethal embryonic mutant phenotypes are rather similar. In homozygous twi or sna embryos invagination of the ventral presumptive mesodermal cells fails to occur, and the resulting embryos are devoid of internal organs. This is very similar to the dominant phenotype described for dl; in the case of dl, however, the effect is a maternal one dependent on the mutant genotype of the female. Second, a synergistic interaction has been found whereby dominant lethality of twi  - or sna-bearing zygotes is observed in embryos derived from heterozygous dl females at high temperature. The temperature sensitivity of this interaction permitted definition of a temperature-sensitive period which is probably that of dl. This was found to extend from approximately 12 hr prior to oviposition to 2— 3 hr of embryogenesis. A zygotic action for the dl gene in addition to the maternal effect was revealed by the finding that extra doses of dl  + in the zygotes can partially rescue the dominant lethality of heterozygous twi embryos derived from heterozygous dl females. Two possible interpretations of the synergism are considered: (1) twi and sna are activated in the embryos as a result of positional signals placed in the egg as a consequence of the functioning of the dl gene during oogenesis and, thus, play a role in embryonic determination. (2) The gene products of dl  + and twi  + (or sna  +) combine to produce a functional molecule that is involved in the specification of dorsoventral pattern in the early embryo.

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