scholarly journals The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

2000 ◽  
Vol 149 (2) ◽  
pp. 397-410 ◽  
Author(s):  
Andreas Wiederkehr ◽  
Sandrine Avaro ◽  
Cristina Prescianotto-Baschong ◽  
Rosine Haguenauer-Tsapis ◽  
Howard Riezman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Trafficking ◽  
Fluorescent Dyes ◽  
Lucifer Yellow ◽  
Endocytic Pathway ◽  
Major Fraction ◽  
Membrane Bound ◽  
Recycling Pathway ◽  
F Box Protein ◽  
Vacuolar Protein

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Δ is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Δ mutant is strongly defective in recycling.

scholarly journals Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

1996 ◽  
Vol 7 (12) ◽  
pp. 1909-1919 ◽  
Author(s):  
M Ziman ◽  
J S Chuang ◽  
R W Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Endocytic Pathway ◽  
Chitin Synthase ◽  
Cell Fractionation ◽  
Cell Wall Polysaccharide ◽  
Chitin Synthesis ◽  
A Cell ◽  
Steady State Levels ◽  
Membrane Bound

In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.

scholarly journals A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages.

1988 ◽  
Vol 107 (3) ◽  
pp. 887-896 ◽  
Author(s):  
T H Steinberg ◽  
J A Swanson ◽  
S C Silverstein
Keyword(s):  
Plasma Membrane ◽  
Lucifer Yellow ◽  
Organic Anion ◽  
Buoyant Density ◽  
Endocytic Pathway ◽  
Cytoplasmic Matrix ◽  
Cytoplasmic Vacuoles ◽  
J774 Cells ◽  
Membrane Bound ◽  
Texas Red

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.

scholarly journals Coordination and Crosstalk between Autophagosome and Multivesicular Body Pathways in Plant Stress Responses

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 119 ◽  
Author(s):  
Mengxue Wang ◽  
Xifeng Li ◽  
Shuwei Luo ◽  
Baofang Fan ◽  
Cheng Zhu ◽  
...  
Keyword(s):  
Stress Responses ◽  
Plant Stress ◽  
Multivesicular Body ◽  
Endocytic Pathway ◽  
Plant Responses ◽  
Hormone Metabolism ◽  
Plant Stress Responses ◽  
Autophagic Degradation ◽  
Membrane Bound ◽  
Vacuolar Protein

In eukaryotic cells, autophagosomes and multivesicular bodies (MVBs) are two closely related partners in the lysosomal/vacuolar protein degradation system. Autophagosomes are double membrane-bound organelles that transport cytoplasmic components, including proteins and organelles for autophagic degradation in the lysosomes/vacuoles. MVBs are single-membrane organelles in the endocytic pathway that contain intraluminal vesicles whose content is either degraded in the lysosomes/vacuoles or recycled to the cell surface. In plants, both autophagosome and MVB pathways play important roles in plant responses to biotic and abiotic stresses. More recent studies have revealed that autophagosomes and MVBs also act together in plant stress responses in a variety of processes, including deployment of defense-related molecules, regulation of cell death, trafficking and degradation of membrane and soluble constituents, and modulation of plant hormone metabolism and signaling. In this review, we discuss these recent findings on the coordination and crosstalk between autophagosome and MVB pathways that contribute to the complex network of plant stress responses.

End13p/Vps4p is required for efficient transport from early to late endosomes in Saccharomyces cerevisiae

2001 ◽  
Vol 114 (10) ◽  
pp. 1935-1947 ◽  
Author(s):  
R. Zahn ◽  
B.J. Stevenson ◽  
S. Schroder-Kohne ◽  
B. Zanolari ◽  
H. Riezman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Dyes ◽  
Membrane Receptors ◽  
Endocytic Pathway ◽  
Aaa Atpase ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Plasma Membrane Receptors ◽  
Vacuolar Protein ◽  
Mutant Cells

end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps(-) phenotype), leading to secretion of newly synthesised vacuolar proteins. Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase. We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p. It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)((Dgr;)) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC). We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole. Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.

scholarly journals Identification and functional characterization of a novel Candida albicans gene CaMNN5 that suppresses the iron-dependent growth defect of Saccharomyces cerevisiae aft1Δ mutant

2005 ◽  
Vol 389 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Chen BAI ◽  
Fong Yee CHAN ◽  
Yue WANG
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Iron Uptake ◽  
Iron Transport ◽  
Lucifer Yellow ◽  
Functional Characterization ◽  
Endocytic Pathway ◽  
Transport Systems ◽  
Growth Defect ◽  
Iron Starvation

In Saccharomyces cerevisiae, the transcription factor Aft1p plays a central role in regulating many genes involved in iron acquisition and utilization. An aft1Δ mutant exhibits severely retarded growth under iron starvation. To identify the functional counterpart of AFT1 in Candida albicans, we transformed a C. albicans genomic DNA library into aft1Δ to isolate genes that could allow the mutant to grow under iron-limiting conditions. In the present paper, we describe the unexpected discovery in this screen of CaMNN5. CaMnn5p is an α-1,2-mannosyltransferease, but its growth-promoting function in iron-limiting conditions does not require this enzymatic activity. Its function is also independent of the high-affinity iron transport systems that are mediated by Ftr1p and Fth1p. We obtained evidence suggesting that CaMnn5p may function along the endocytic pathway, because it cannot promote the growth of end4Δ and vps4Δ mutants, where the endocytic pathway is blocked at an early and late step respectively. Neither can it promote the growth of a fth1Δ smf3Δ mutant, where the vacuole–cytosol iron transport is blocked. Expression of CaMNN5 in S. cerevisiae specifically enhances an endocytosis-dependent mechanism of iron uptake without increasing the uptake of Lucifer Yellow, a marker for fluid-phase endocytosis. CaMnn5p contains three putative Lys-Glu-Xaa-Xaa-Glu iron-binding sites and co-immunoprecipitates with 55Fe. We propose that CaMnn5p promotes iron uptake and usage along the endocytosis pathway under iron-limiting conditions, a novel function that might have evolved in C. albicans.

scholarly journals Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast.

1994 ◽  
Vol 125 (2) ◽  
pp. 283-298 ◽  
Author(s):  
B Singer-Krüger ◽  
H Stenmark ◽  
A Düsterhöft ◽  
P Philippsen ◽  
J S Yoo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Lucifer Yellow ◽  
Protein Sorting ◽  
Personal Communication ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Phenotypic Characterization ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.

scholarly journals Deubiquitination Step in the Endocytic Pathway of Yeast Plasma Membrane Proteins: Crucial Role of Doa4p Ubiquitin Isopeptidase

2001 ◽  
Vol 21 (14) ◽  
pp. 4482-4494 ◽  
Author(s):  
S. Dupré ◽  
R. Haguenauer-Tsapis
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Degradation Products ◽  
Endocytic Pathway ◽  
Plasma Membrane Proteins ◽  
General Role ◽  
Yeast Plasma Membrane ◽  
Membrane Bound ◽  
Vacuolar Protein

ABSTRACT The Fur4p uracil permease, like most yeast plasma membrane proteins, undergoes ubiquitin-dependent endocytosis and is then targeted to the vacuole (equivalent to the mammalian lysosome) for degradation. The cell surface ubiquitination of Fur4p is mediated by the essential Rsp5p ubiquitin ligase. Ubiquitination of Fur4p occurs on two target lysines, which receive two ubiquitin moieties linked through ubiquitin Lys63, a type of linkage (termed UbK63) different from that involved in proteasome recognition. We report that pep4cells deficient for vacuolar protease activities accumulate vacuolar unubiquitinated Fur4p. In contrast, pep4 cells lacking the Doa4p ubiquitin isopeptidase accumulate ubiquitin-conjugated Fur4p. These data suggest that Fur4p undergoes Doa4p-dependent deubiquitination prior to vacuolar degradation. Compared topep4 cells, pep4 doa4 cells have huge amounts of membrane-bound ubiquitin conjugates. This indicates that Doa4p plays a general role in the deubiquitination of membrane-bound proteins, as suggested by reports describing the suppression of somedoa4 phenotypes in endocytosis and vacuolar protein sorting mutants. Some of the small ubiquitin-linked peptides that are a hallmark of Doa4 deficiency are not present in rsp5 mutant cells or after overproduction of a variant ubiquitin modified at Lys 63 (UbK63R). These data suggest that the corresponding peptides are degradation products of Rsp5p substrates and probably of ubiquitin conjugates carrying UbK63 linkages. Doa4p thus appears to be involved in the deubiquitination of endocytosed plasma membrane proteins, some of them carrying UbK63 linkages.

scholarly journals Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells

2021 ◽  
Vol 22 (15) ◽  
pp. 8165
Author(s):  
Amanda Chantziou ◽  
Kostas Theodorakis ◽  
Hara Polioudaki ◽  
Eelco de Bree ◽  
Marilena Kampa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Plasma Membrane ◽  
Subcellular Localization ◽  
Cancer Cells ◽  
Membrane Trafficking ◽  
Breast Cancer Cells ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Wild Type ◽  
Mcf 7

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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