Involvement of Ypt7p, a small GTPase, in traffic from late endosome to the vacuole in yeast

1993 ◽  
Vol 106 (3) ◽  
pp. 823-830 ◽  
Author(s):  
F. Schimmoller ◽  
H. Riezman
Keyword(s):  
Membrane System ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Independent Evidence ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Alpha Factor ◽  
Regulation Of Transport ◽  
Vacuole Biogenesis

The YPT7 gene encodes the Saccharomyces cerevisiae homolog of mammalian rab7 protein. Data obtained from studies on a delta ypt7 mutant suggested that Ypt7p is involved in the endocytic pathway in yeast (Wichmann et al., Cell 71, 1131–1142, 1992). We report here that endocytosed pheromone alpha-factor accumulates in late endosomes in delta ypt7 cells, indicating that Ypt7p is involved in the regulation of transport steps from late endosomes to the vacuole. We also show that alpha-factor can be degraded in a PEP4-dependent manner in a prevacuolar/endosomal compartment in delta ypt7 cells, providing independent evidence that the pathways of vacuole biogenesis and endocytosis in yeast may intersect in the endosomal membrane system.

scholarly journals Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication

2004 ◽  
Vol 15 (6) ◽  
pp. 2954-2964 ◽  
Author(s):  
Marije Marsman ◽  
Ingrid Jordens ◽  
Coen Kuijl ◽  
Lennert Janssen ◽  
Jacques Neefjes
Keyword(s):  
Hela Cells ◽  
Small Gtpase ◽  
Vesicle Transport ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Intracellular Replication ◽  
Motor Complex ◽  
Late Endosomes ◽  
Membrane Bound ◽  
Gtpase Cycle

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.

Yeast Ypt51p and mammalian Rab5: counterparts with similar function in the early endocytic pathway

1995 ◽  
Vol 108 (11) ◽  
pp. 3509-3521 ◽  
Author(s):  
B. Singer-Kruger ◽  
H. Stenmark ◽  
M. Zerial
Keyword(s):  
Small Gtpase ◽  
Endocytic Pathway ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Cell Fractionation ◽  
Animal Cells ◽  
Early Endosomes ◽  
Fractionation Analysis ◽  
Alpha Factor ◽  
Vacuolar Protein

Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since alpha-factor accumulated in an early endocytic intermediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi compartment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for alpha-factor receptor and for carboxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.

scholarly journals Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

2021 ◽  
Author(s):  
Paula Ortega Gonzalez ◽  
Gwen M Taylor ◽  
Rohit Jangra ◽  
Raquel Tenorio Vela ◽  
Isabel Fernandez de Castro Martin ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Cholesterol Homeostasis ◽  
Endocytic Pathway ◽  
Target Cells ◽  
Genome Replication ◽  
Reovirus Infection ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Mammalian Orthoreovirus ◽  
Niemann Pick

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.

scholarly journals Identification of a new, Rab14-dependent, endo-lysosomal pathway

2021 ◽  
Author(s):  
Evgeniya Trofimenko ◽  
Yuta Homma ◽  
Mitsunori Fukuda ◽  
Christian Widmann
Keyword(s):  
Common Ancestor ◽  
Cell Penetrating Peptides ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Last Eukaryotic Common Ancestor ◽  
Surrounding Environment ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Pathway ◽  
Dependent Pathway

Cells can endocytose material from the surrounding environment. Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Several endocytosis pathways have been described (e.g. clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway). Besides possible recycling routes to the plasma membrane and various organelles, these pathways all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs) used in research and clinic, we have discovered a second endocytic pathway that moves material to late endosomes/lysosomes and that is fully independent of Rab5 and Rab7 but requires the Rab14 protein. This newly identified pathway differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already and is not affected by a series of compounds that inhibit the Rab5-dependent pathway. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins. Rab14 is expressed by the last eukaryotic common ancestor. The Rab14-dependent pathway may therefore correspond to a primordial endosomal pathway taken by cationic cargos.

The role of calcium and other ions in sorting and delivery in the late endocytic pathway

2007 ◽  
Vol 35 (5) ◽  
pp. 1088-1091 ◽  
Author(s):  
J.P. Luzio ◽  
N.A. Bright ◽  
P.R. Pryor
Keyword(s):  
Mammalian Cells ◽  
Ionic Composition ◽  
Endocytic Pathway ◽  
Lysosomal Storage ◽  
Acid Hydrolases ◽  
Gene Encoding ◽  
Endosomal Membrane ◽  
Type Iv ◽  
Late Endosomes ◽  
The Many

The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain many luminal vesicles and are often referred to as MVBs (multivesicular bodies). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. The luminal ionic composition of organelles in the endocytic pathway is of considerable importance both in the trafficking of endocytosed ligands and in the membrane fusion events. In particular, H+ ions play a role in sorting processes and providing an appropriate environment for the action of lysosomal acid hydrolases. Na+/H+ exchangers in the endosomal membrane have been implicated in the formation of MVBs and sorting into luminal vesicles. Ca2+ ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca2+ in traffic through the late endocytic pathway, mutations in the gene encoding mucolipin-1, a lysosomal non-specific cation channel, result in abnormalities in lipid traffic and are associated with the autosomal recessive lysosomal storage disease MLIV (mucolipidosis type IV).

scholarly journals Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein-mediated basipetal movement

2012 ◽  
Vol 23 (10) ◽  
pp. 1889-1901 ◽  
Author(s):  
Juan F. Abenza ◽  
Antonio Galindo ◽  
Mario Pinar ◽  
Areti Pantazopoulou ◽  
Vivian de los Ríos ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Multivesicular Body ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Long Distance ◽  
Genetic Studies ◽  
Late Endosomes ◽  
Early Endosomes ◽  
A Minor ◽  
Long Distance Movement

We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabSRab7 mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41–dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabSRab7 are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabSRab7, early endosomal Rab5s—RabA and RabB—reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabSRab7 is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabSRab7 detectable in the same—frequently the less motile—structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.

scholarly journals Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

2016 ◽  
Vol 27 (2) ◽  
pp. 334-348 ◽  
Author(s):  
Muralidharan Mani ◽  
Unn Hwa Lee ◽  
Nal Ae Yoon ◽  
Hyo Jeong Kim ◽  
Myoung Seok Ko ◽  
...  
Keyword(s):  
Binding Protein ◽  
Ectopic Expression ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Gtp Binding Protein ◽  
Developmentally Regulated ◽  
Gtp Binding ◽  
Phosphatidylinositol 3

The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling.

scholarly journals Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

2019 ◽  
Vol 77 (14) ◽  
pp. 2839-2857 ◽  
Author(s):  
Elsa Meneses-Salas ◽  
Ana García-Melero ◽  
Kristiina Kanerva ◽  
Patricia Blanco-Muñoz ◽  
Frederic Morales-Paytuvi ◽  
...  
Keyword(s):  
Late Endosome ◽  
Dependent Manner ◽  
Lipid Transfer ◽  
Cholesterol Accumulation ◽  
Membrane Contact Sites ◽  
Late Endosomes ◽  
Membrane Contact ◽  
Domain 3 ◽  
Niemann Pick ◽  
Mutant Cells

Abstract Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

scholarly journals Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol

2021 ◽  
Author(s):  
Juan Carlos Polanco ◽  
Gabriel Rhys Hand ◽  
Adam Briner ◽  
Chuanzhou Li ◽  
Jürgen Götz
Keyword(s):  
Critical Role ◽  
Membrane Integrity ◽  
Lysosomal Degradation ◽  
Escape Route ◽  
Cellular Invasion ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Endosomal Pathway ◽  
Tau Aggregation

AbstractThe microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

scholarly journals TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans

2010 ◽  
Vol 21 (13) ◽  
pp. 2285-2296 ◽  
Author(s):  
Laëtitia Chotard ◽  
Ashwini K. Mishra ◽  
Marc-André Sylvain ◽  
Simon Tuck ◽  
David G. Lambright ◽  
...  
Keyword(s):  
Rab Gtpase ◽  
Endosomal Trafficking ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Late Endosomes ◽  
Arginine Finger ◽  
Endosome Maturation ◽  
Hops Complex

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

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