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.

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 The Yeast vps Class E Mutants: The Beginning of the Molecular Genetic Analysis of Multivesicular Body Biogenesis

2010 ◽  
Vol 21 (23) ◽  
pp. 4057-4060 ◽  
Author(s):  
Emily M. Coonrod ◽  
Tom H. Stevens
Keyword(s):  
Membrane Proteins ◽  
Molecular Genetic ◽  
Multivesicular Body ◽  
Molecular Genetic Analysis ◽  
Vacuolar Membrane ◽  
Multivesicular Bodies ◽  
Golgi Membrane ◽  
Cargo Proteins ◽  
Vacuolar Protein ◽  
Class E

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this “class E compartment” contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

scholarly journals Structure and function of human Vps20 and Snf7 proteins

2004 ◽  
Vol 377 (3) ◽  
pp. 693-700 ◽  
Author(s):  
Jeremy W. PECK ◽  
Emma T. BOWDEN ◽  
Peter D. BURBELO
Keyword(s):  
Protein Interactions ◽  
Osmotic Shock ◽  
Multivesicular Body ◽  
Protein Protein Interactions ◽  
Interacting Protein ◽  
Endosomal Membrane ◽  
Genomic Studies ◽  
Vacuolar Protein ◽  
And Function

Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function. In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans. Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant. In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles. Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting. To explore possible protein–protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function. Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 [ALG-2 (apoptosis-linked gene 2) interacting protein 1], a mammalian Bro1p [BCK1 (bypass of C kinase)-like resistance to osmotic shock]-containing protein involved in cellular vacuolization and apoptosis. Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an α-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1. Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.

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.

scholarly journals A structural model of the active ribosome-bound membrane protein insertase YidC

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Stephan Wickles ◽  
Abhishek Singharoy ◽  
Jessica Andreani ◽  
Stefan Seemayer ◽  
Lukas Bischoff ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Structural Model ◽  
Cytoplasmic Membrane ◽  
Membrane Surface ◽  
Single Copy ◽  
Protein Insertion ◽  
Versus Protein ◽  
Membrane Protein Insertion ◽  
Translational Mode ◽  
Dynamics Simulations

The integration of most membrane proteins into the cytoplasmic membrane of bacteria occurs co-translationally. The universally conserved YidC protein mediates this process either individually as a membrane protein insertase, or in concert with the SecY complex. Here, we present a structural model of YidC based on evolutionary co-variation analysis, lipid-versus-protein-exposure and molecular dynamics simulations. The model suggests a distinctive arrangement of the conserved five transmembrane domains and a helical hairpin between transmembrane segment 2 (TM2) and TM3 on the cytoplasmic membrane surface. The model was used for docking into a cryo-electron microscopy reconstruction of a translating YidC-ribosome complex carrying the YidC substrate FOc. This structure reveals how a single copy of YidC interacts with the ribosome at the ribosomal tunnel exit and identifies a site for membrane protein insertion at the YidC protein-lipid interface. Together, these data suggest a mechanism for the co-translational mode of YidC-mediated membrane protein insertion.

scholarly journals Efficient Cargo Sorting by ESCRT-I and the Subsequent Release of ESCRT-I from Multivesicular Bodies Requires the Subunit Mvb12

2007 ◽  
Vol 18 (2) ◽  
pp. 636-645 ◽  
Author(s):  
Matt Curtiss ◽  
Charles Jones ◽  
Markus Babst
Keyword(s):  
Protein Complex ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Rapid Degradation ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Complex Functions ◽  
Vacuolar Protein ◽  
Cargo Sorting

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.

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 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 Dual Sorting of the Saccharomyces cerevisiae Vacuolar Protein Sna4p

2009 ◽  
Vol 8 (3) ◽  
pp. 278-286 ◽  
Author(s):  
W. Pokrzywa ◽  
B. Guerriat ◽  
J. Dodzian ◽  
P. Morsomme
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alkaline Phosphatase ◽  
Membrane Protein ◽  
Ubiquitin Ligase ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Vacuolar Membrane ◽  
Multivesicular Bodies ◽  
Small Family ◽  
Vacuolar Protein

ABSTRACT Sna4p, a vacuolar membrane protein, belongs to a small family of proteins conserved in plants and fungi. It is transported to the vacuolar membrane via the alkaline phosphatase (ALP) pathway, which bypasses the multivesicular bodies (MVBs). Here, we show that transfer of Sna4p by the ALP route involves the AP-3 adaptor protein complex, which binds to an acidic dileucine sorting signal in the cytoplasmic region of Sna4p. In addition, Sna4p can use the MVB pathway by using a PPPY motif, which is involved in the interaction with ubiquitin ligase Rsp5p. Deletion or mutation of the Sna4p PPPY motif or a low level of Rsp5p inhibits the entrance of Sna4p into MVBs. Sna4p is polyubiquitylated on its only lysine, and Sna4p lacking this lysine shows defective MVB sorting. These data indicate that Sna4p has two functional motifs, one for interaction with the AP-3 complex, followed by entry into the ALP pathway, and one for binding Rsp5p, which directs the protein to the MVB pathway. The presence of these two motifs allows Sna4p to localize to both the vacuolar membrane and the lumen.

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