scholarly journals Import into and Degradation of Cytosolic Proteins by Isolated Yeast Vacuoles

1999 ◽  
Vol 10 (9) ◽  
pp. 2879-2889 ◽  
Author(s):  
Martin Horst ◽  
Erwin C. Knecht ◽  
Peter V. Schu
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Protein Import ◽  
Degradation Pathway ◽  
Specific Protein ◽  
Transport Pathway ◽  
Cytosolic Proteins ◽  
Hsp70 Family ◽  
Vacuolar Protein ◽  
Shock Proteins

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.

scholarly journals Vacuole Biogenesis in Saccharomyces cerevisiae: Protein Transport Pathways to the Yeast Vacuole

1998 ◽  
Vol 62 (1) ◽  
pp. 230-247 ◽  
Author(s):  
Nia J. Bryant ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Delivery ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Transport Pathway ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transport Pathways ◽  
Yeast Vacuole ◽  
Lysosome Biogenesis ◽  
Vacuolar Protein

SUMMARY Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic. This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane. Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy. Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole. This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S. cerevisiae.

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 An arf1Δ Synthetic Lethal Screen Identifies a New Clathrin Heavy Chain Conditional Allele That Perturbs Vacuolar Protein Transport in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (2) ◽  
pp. 577-589 ◽  
Author(s):  
Chih-Ying Chen ◽  
Todd R Graham
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Protein Transport ◽  
Genetic Interaction ◽  
Synthetic Lethality ◽  
Carboxypeptidase Y ◽  
Coat Proteins ◽  
Transport Pathway ◽  
Clathrin Heavy Chain ◽  
Vacuolar Protein

Abstract ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Δ allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Δ). Most of the swa mutants exhibit phenotypes comparable to arf1Δ mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3′ end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.

scholarly journals Involvement of 70-kD heat-shock proteins in peroxisomal import.

1994 ◽  
Vol 125 (5) ◽  
pp. 1037-1046 ◽  
Author(s):  
P A Walton ◽  
M Wendland ◽  
S Subramani ◽  
R A Rachubinski ◽  
W J Welch
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Protein Import ◽  
Peroxisomal Membrane ◽  
Permeabilized Cell ◽  
Hsp70 Family ◽  
Cell Biol ◽  
Exogenous Hsp70 ◽  
Shock Proteins ◽  
Liver Peroxisomes

This report describes the involvement of 70-kD heat-shock proteins (hsp70) in the import of proteins into mammalian peroxisomes. Employing a microinjection-based assay (Walton, P. A., S. J. Gould, J. R. Feramisco, and S. Subramani. 1992. Mol. Cell Biol. 12:531-541), we demonstrate that proteins of the hsp70 family were associated with proteins being imported into the peroxisomal matrix. Import of peroxisomal proteins could be inhibited by coinjection of antibodies directed against the constitutive hsp70 proteins (hsp73). In a permeabilized-cell assay (Wendland and Subramani. 1993. J. Cell Biol. 120:675-685), antibodies directed against hsp70 proteins were shown to inhibit peroxisomal protein import. Inhibition could be overcome by the addition of exogenous hsp70 proteins. Purified rat liver peroxisomes were shown to have associated hsp70 proteins. The amount of associated hsp70 was increased under conditions of peroxisomal proliferation. Furthermore, proteinase protection assays indicated that the hsp70 molecules were located on the outside of the peroxisomal membrane. Finally, the process of heat-shocking cells resulted in a considerable delay in the import of peroxisomal proteins. Taken together, these results indicate that heat-shock proteins of the cytoplasmic hsp70 family are involved in the import of peroxisomal proteins.

scholarly journals DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii63-ii63
Author(s):  
Lakshmi Bollu ◽  
Derek Wainwright ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Time Course ◽  
Immune Cell ◽  
Enzyme Inhibitors ◽  
Tumor Development ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Specific Protein ◽  
Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

scholarly journals The RNA Degradation Pathway Regulates the Function of GAS5 a Non-Coding RNA in Mammalian Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. e55684 ◽  
Author(s):  
Hidenori Tani ◽  
Masaki Torimura ◽  
Nobuyoshi Akimitsu
Keyword(s):  
Mammalian Cells ◽  
Rna Degradation ◽  
Degradation Pathway ◽  
Non Coding Rna

Evidence for an ER to Golgi to chloroplast protein transport pathway

2006 ◽  
Vol 16 (8) ◽  
pp. 385-387 ◽  
Author(s):  
Resmi N. Radhamony ◽  
Steven M. Theg
Keyword(s):  
Protein Transport ◽  
Transport Pathway ◽  
Chloroplast Protein

Herpes simplex virus type 2 UL14 gene product has heat shock protein(HSP)-like functions

2002 ◽  
Vol 115 (12) ◽  
pp. 2517-2527
Author(s):  
Yohei Yamauchi ◽  
Kaoru Wada ◽  
Fumi Goshima ◽  
Tohru Daikoku ◽  
Kenzo Ohtsuka ◽  
...  
Keyword(s):  
Heat Shock ◽  
Nuclear Translocation ◽  
Osmotic Shock ◽  
Minor Component ◽  
Firefly Luciferase ◽  
Hsp70 Family ◽  
Arginine Residues ◽  
Simplex Virus ◽  
A Minor ◽  
Shock Proteins

The HSV-2 UL14 gene encodes a 32 kDa protein that is a minor component of the viral tegument. The protein relocates other viral proteins such as VP26 and UL33 protein into the nuclei of transiently coexpressing cells(Yamauchi et al., 2001). We found that the protein shared some characteristics of heat shock proteins(HSPs) or molecular chaperones, such as nuclear translocation upon heat shock,ATP deprivation and osmotic shock. Interestingly, a significant homology over a stretch of 15 amino acids was found between an N-terminal region of HSV UL14 protein and the substrate-binding domain of Hsp70 family proteins. Two arginine residues in this region were important for nuclear translocation of VP26. In addition, overexpression of UL14 protein increased the activity of coexpressed firefly luciferase, which suggested that the protein functioned in the folding of newly synthesized luciferase. We thus conclude that UL14 protein can act as a chaperone-like protein in a singly expressed state.

Genetic analysis of intracellular aminoglycerophospholipid traffic

2004 ◽  
Vol 82 (1) ◽  
pp. 156-169 ◽  
Author(s):  
Dennis R Voelker
Keyword(s):  
Genetic Analysis ◽  
Mammalian Cells ◽  
Molecular Genetic ◽  
Family Members ◽  
Molecular Genetic Analysis ◽  
Transport Processes ◽  
Membrane Biogenesis ◽  
Independent Manner ◽  
Multiple Genes ◽  
Vacuolar Protein

Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis. Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions. Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination. In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes. Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase. Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole. In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members. The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner. The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.Key words: lipid transport, lipid sorting, membrane biogenesis, organelles, flippase.

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