scholarly journals The penta-EF-hand protein ALG-2 interacts directly with the ESCRT-I component TSG101, and Ca2+-dependently co-localizes to aberrant endosomes with dominant-negative AAA ATPase SKD1/Vps4B

2005 ◽  
Vol 391 (3) ◽  
pp. 677-685 ◽  
Author(s):  
Keiichi Katoh ◽  
Hidenori Suzuki ◽  
Yoshinori Terasawa ◽  
Takako Mizuno ◽  
Jiro Yasuda ◽  
...  
Keyword(s):  
Hela Cells ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Positive Interaction ◽  
Interacting Protein ◽  
Body Protein ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Ef Hand ◽  
Vacuolar Protein

ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIP1 (ALG-2-interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport I) and ESCRT-III respectively. In the present study, we investigated the association between ALG-2 and ESCRT-I. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] were shown to associate with GST–ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101. Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpressed a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD1/Vps4B (GFP–SKD1E235Q), ALG-2 exhibited a punctate distribution at the perinuclear area and co-localized with GFP–SKD1E235Q to aberrant endosomes. This punctate distribution of ALG-2 was markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 did not co-localize with GFP–SKD1E235Q. Our findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix.

scholarly journals CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway

2006 ◽  
Vol 400 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Mio Horii ◽  
Hideki Shibata ◽  
Ryota Kobayashi ◽  
Keiichi Katoh ◽  
Chiharu Yorikawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Amino Acid Residues ◽  
Related Protein ◽  
Positive Interaction ◽  
Leukaemia Virus ◽  
Gag Protein ◽  
Endosomal Sorting ◽  
Aaa Atpase

All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1–CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG–CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG–CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b–GFP or GFP–Vps4BE235Q, a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP–CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP–CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting pathway.

scholarly journals Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting

2005 ◽  
Vol 387 (1) ◽  
pp. 17-26 ◽  
Author(s):  
Chiharu YORIKAWA ◽  
Hideki SHIBATA ◽  
Satoshi WAGURI ◽  
Kazumi HATTA ◽  
Mio HORII ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Membrane Surface ◽  
Consensus Sequence ◽  
Multivesicular Body ◽  
Body Protein ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Vacuolar Protein ◽  
Cargo Sorting

CHMP6 (charged multivesicular body protein 6) is a human orthologue of yeast Vps (vacuolar protein sorting) 20, a component of ESCRT (endosomal sorting complex required for transport)-III. Various CHMP6 orthologues in organisms ranging from yeast to humans contain the N-myristoylation consensus sequence at each N-terminus. Metabolic labelling of HEK-293 (human embryonic kidney) cells showed the incorporation of [3H]myristate into CHMP6 fused C-terminally to GFP (green fluorescent protein) (CHMP6–GFP). Interactions of CHMP6 with another ESCRT-III component CHMP4b/Shax [Snf7 (sucrose non-fermenting 7) homologue associated with Alix] 1, one of three paralogues of human Vps32/Snf7, and with EAP20 (ELL-associated protein 20), a human counterpart of yeast Vps25 and component of ESCRT-II, were observed by co-immunoprecipitation of epitope-tagged proteins expressed in HEK-293 cells. The in vitro pull-down assays using their recombinant proteins purified from Escherichia coli demonstrated direct physical interactions which were mediated by the N-terminal basic half of CHMP6. Overexpressed CHMP6-GFP in HeLa cells exhibited a punctate distribution throughout the cytoplasm especially in the perinuclear area, as revealed by fluorescence microscopic analysis. Accumulation of LBPA (lysobisphosphatidic acid), a major phospholipid in internal vesicles of an MVB (multivesicular body), was observed in the CHMP6–GFP-localizing area. FLAG-tagged EAP20 distributed diffusely, but exhibited a punctate distribution on co-expression with CHMP6–GFP. Overexpression of CHMP6–GFP caused reduction of transferrin receptors on the plasma membrane surface, but caused their accumulation in the cytoplasm. Ubiquitinated proteins and endocytosed EGF continuously accumulated in CHMP6–GFP-expressing cells. These results suggest that CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes and regulates cargo sorting.

Structure and function of ESCRT-III

2009 ◽  
Vol 37 (1) ◽  
pp. 156-160 ◽  
Author(s):  
Suman Lata ◽  
Guy Schoehn ◽  
Julianna Solomons ◽  
Ricardo Pires ◽  
Heinrich G. Göttlinger ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Tubular Structures ◽  
Body Protein ◽  
Enveloped Viruses ◽  
Endosomal Sorting ◽  
Vacuolar Protein ◽  
And Function ◽  
Endosomal Vesicles

ESCRT-III (endosomal sorting complex required for transport III) is required for the formation and abscission of intraluminal endosomal vesicles, which gives rise to multivesicular bodies, budding of some enveloped viruses and cytokinesis. ESCRT-III is composed of 11 members in humans, which, except for one, correspond to the six ESCRT-III-like proteins in yeast. At least CHMP (charged multivesicular body protein) 2A and CHMP3 assemble into helical tubular structures that provide a platform for membrane interaction and VPS (vacuolar protein sorting) 4-catalysed effects leading to disassembly of ESCRT-III CHMP2A–CHMP3 polymers in vitro. Progress towards the understanding of the structures and function of ESCRT-III, its activation, its regulation by accessory factors and its role in abscission of membrane enveloped structures in concert with VPS4 are discussed.

scholarly journals Golgi-bound Rab34 Is a Novel Member of the Secretory Pathway

2007 ◽  
Vol 18 (12) ◽  
pp. 4762-4771 ◽  
Author(s):  
Neil M. Goldenberg ◽  
Sergio Grinstein ◽  
Mel Silverman
Keyword(s):  
Hela Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Golgi Stack ◽  
Green Fluorescent ◽  
Intracellular Vesicle Transport

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

scholarly journals Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation

2009 ◽  
Vol 83 (20) ◽  
pp. 10797-10807 ◽  
Author(s):  
Ritesh Tandon ◽  
David P. AuCoin ◽  
Edward S. Mocarski
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Body Protein ◽  
Escrt Machinery ◽  
Hcmv Replication ◽  
Viral Maturation ◽  
Vacuolar Protein

ABSTRACT The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9:2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4DN) in HCMV replication. Vps4DN specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1DN) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101DN) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4DN or CHMP1DN blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.

The CHMP4b- and Src-docking sites in the Bro1 domain are autoinhibited in the native state of Alix

2009 ◽  
Vol 418 (2) ◽  
pp. 277-284 ◽  
Author(s):  
Xi Zhou ◽  
Shujuan Pan ◽  
Le Sun ◽  
Joe Corvera ◽  
Yu-Chen Lee ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Human Embryonic Kidney ◽  
Native State ◽  
Interacting Protein ◽  
Body Protein ◽  
Linked Gene ◽  
Endosomal Sorting ◽  
Protein X ◽  
Membrane Bound ◽  
Docking Sites

The Bro1 domain of Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], which plays important roles in endosomal sorting and multiple ESCRT (endosomal sorting complex required for transport)-linked processes, contains the docking sites for the ESCRT-III component CHMP4b (charged multivesicular body protein 4b) and the regulatory tyrosine kinase, Src. Although the structural bases for these docking sites have been defined by crystallography studies, it has not been determined whether these sites are available in the native state of Alix. In the present study, we demonstrate that these two docking sites are unavailable in recombinant Alix under native conditions and that their availabilities can be induced by detergents. In HEK (human embryonic kidney)-293 cell lysates, these two docking sites are not available in cytosolic Alix, but are available in membrane-bound Alix. These findings show that the native state of Alix does not have a functional Bro1 domain and predict that Alix's involvement in endosomal sorting and other ESCRT-linked processes requires an activation step that relieves the autoinhibition of the Bro1 domain.

scholarly journals Regulation of Vps4 ATPase activity by ESCRT-III

2009 ◽  
Vol 37 (1) ◽  
pp. 143-145 ◽  
Author(s):  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
David J. Katzmann
Keyword(s):  
Atpase Activity ◽  
Multivesicular Body ◽  
Critical Function ◽  
Body Formation ◽  
Temporal Regulation ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Endosomal Membrane ◽  
Endosomal Membranes ◽  
Vacuolar Protein

MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.

scholarly journals Structural Insights into AQP2 Targeting to Multivesicular Bodies

2019 ◽  
Vol 20 (21) ◽  
pp. 5351
Author(s):  
Jennifer Virginia Roche ◽  
Veronika Nesverova ◽  
Caroline Olsson ◽  
Peter MT Deen ◽  
Susanna Törnroth-Horsefield
Keyword(s):  
Membrane Protein ◽  
Structural Model ◽  
Collecting Duct ◽  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Interacting Protein ◽  
Body Protein ◽  
Protein Insertion ◽  
Vacuolar Protein ◽  
Structural Insights

Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.

scholarly journals TSG101/Mammalian VPS23 and Mammalian VPS28 Interact Directly and Are Recruited to VPS4-induced Endosomes

2000 ◽  
Vol 276 (15) ◽  
pp. 11735-11742 ◽  
Author(s):  
Naomi Bishop ◽  
Philip Woodman
Keyword(s):  
Structural Information ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Dominant Negative Mutant ◽  
Multivesicular Bodies ◽  
Multiprotein Complex ◽  
Endosomal Sorting ◽  
Form Part ◽  
Vacuolar Protein ◽  
Class E

Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.

A dominant-negative ESCRT-III protein perturbs cytokinesis and trafficking to lysosomes

2008 ◽  
Vol 411 (2) ◽  
pp. 233-239 ◽  
Author(s):  
Joseph D. Dukes ◽  
Judith D. Richardson ◽  
Ruth Simmons ◽  
Paul Whitley
Keyword(s):  
Membrane Trafficking ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Eukaryotic Cells ◽  
Body Protein ◽  
Recycling Endosomes ◽  
Protein Subunits ◽  
Autoinhibitory Domain ◽  
Endosomal Sorting ◽  
A Subunit

In eukaryotic cells, the completion of cytokinesis is dependent on membrane trafficking events to deliver membrane to the site of abscission. Golgi and recycling endosomal-derived proteins are required for the terminal stages of cytokinesis. Recently, protein subunits of the ESCRT (endosomal sorting complexes required for transport) that are normally involved in late endosome to lysosome trafficking have also been implicated in abscission. Here, we report that a subunit, CHMP3 (charged multivesicular body protein-3), of ESCRT-III localizes at the midbody. Deletion of the C-terminal autoinhibitory domain of CHMP3 inhibits cytokinesis. At the midbody, CHMP3 does not co-localize with Rab11, suggesting that it is not present on recycling endosomes. These results combined provide compelling evidence that proteins involved in late endosomal function are necessary for the end stages of cytokinesis.

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