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PubMed2006

CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis.

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.

Activin Receptors, Type I↗

PubMed2003

Cdc50p, a conserved endosomal membrane protein, controls polarized growth in Saccharomyces cerevisiae.

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.

Actins↗

PubMed2003

Endosomal dynamics of Met determine signaling output.

Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place (). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5-10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.

Acetylcysteine↗

PubMed2003

STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif.

Conjugation with ubiquitin acts as a sorting signal for proteins in the endocytic and biosynthetic pathways at the endosome. Signal-transducing adaptor molecule (STAM) proteins, STAM1 and STAM2, are associated with hepatocyte growth factor-regulated substrate (Hrs) but their function remains unknown. Herein, we show that STAM proteins bind ubiquitin and ubiquitinated proteins and that the tandemly located VHS (Vps27/Hrs/STAM) domain and ubiquitin-interacting motif serve as the binding site(s). STAM proteins colocalize with Hrs on the early endosome. Overexpression of STAM proteins, but not their mutants lacking the ubiquitin-binding activity, causes the accumulation of ubiquitinated proteins and ligand-activated epidermal growth factor receptor on the early endosome. These results suggest that through interaction with ubiquitinated cargo proteins on the early endosome via the VHS domain and ubiquitin-interacting motif, STAM proteins participate in the sorting of cargo proteins for trafficking to the lysosome.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation.

The yeast uracil permease, Fur4p, is downregulated by uracil, which is toxic to cells with high permease activity. Uracil promotes cell surface Rsp5p-dependent ubiquitylation of the permease, signaling its endocytosis and further vacuolar degradation. We show here that uracil also triggers the direct routing of its cognate permease from the Golgi apparatus to the endosomal system for degradation, without passage via the plasma membrane. This early sorting was not observed for a variant permease with a much lower affinity for uracil, suggesting that uracil binding is the signal for the diverted pathway. The FUI1-encoded uridine permease is similarly sorted for early vacuolar degradation in cells exposed to a toxic level of uridine uptake. Membrane proteins destined for vacuolar degradation require sorting at the endosome level to the intraluminal vesicles of the multivesicular bodies. In cells with low levels of Rsp5p, Fur4p can be still diverted from the Golgi apparatus but does not reach the vacuolar lumen, being instead missorted to the vacuolar membrane. Correct luminal delivery is restored by the biosynthetic addition of a single ubiquitin, suggesting that the ubiquitylation of Fur4p serves as a specific signal for sorting to the luminal vesicles of the multivesicular bodies. A fused ubiquitin is also able to sort some Fur4p from the Golgi to the degradative pathway in the absence of added uracil but the low efficiency of this sorting indicates that ubiquitin does not itself act as a dominant signal for Golgi-to-endosome trafficking. Our results are consistent with a model in which the binding of intracellular uracil to the permease signals its sorting from the Golgi apparatus and subsequent ubiquitylation ensures its delivery to the vacuolar lumen.

Cell Membrane↗

PubMed2003

Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S.

The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole. Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway. Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2. A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS. However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants. We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2. However, our analyses reveal that fab1 mutants do not exhibit a defect in ubiquitination of pCPS. Thus, both Rsp5 and Fab1 play distinct and essential roles in the targeting of biosynthetic MVB cargoes. However, whereas Rsp5 seems to be responsible for cargo ubiquitination, the precise role for Fab1 remains to be elucidated.

Carboxypeptidases↗

PubMed2004

Essential role for the myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p in regulation of phosphatidylinositol 3-phosphate in yeast.

The requirement of Vps34p, the sole phosphatidylinositol (PI) 3-kinase in Saccharomyces cerevisiae, for protein sorting to the vacuole in yeast has exemplified the essential role for phosphoinositides, phosphorylated derivatives of PI, in membrane trafficking. To better understand mechanisms that regulate PI 3-phosphate [PI(3)P]-mediated signaling, the role of the yeast myotubularin-related PI(3)P phosphatase Ymr1p was investigated. We found that Ymr1p and the synaptojanin-like phosphatase Sjl3p function as key regulators of the localization and levels of PI(3)P. Our data indicated that the ymr1Delta sjl3Delta double mutant aberrantly accumulated PI(3)P and demonstrated a steady-state redistribution of this lipid that leads to enrichment on the vacuolar membrane. This resulted in vacuole protein sorting defects, vacuolar fragmentation, and the misregulation of PI(3)P-specific effectors. Triple deletion of YMR1, SJL2, and SJL3 was lethal, suggesting an essential requirement for phosphatase-mediated PI(3)P regulation. Consistent with this, growth was restored to a ymr1Delta sjl2Delta sjl3Delta triple mutant by a PI(3)P-targeted Sac1p domain chimera (GFP-Sac1DeltaC-FYVE(EEA1)) that returned PI(3)P to levels comparable with wild-type cells. Together, this study demonstrated that Ymr1p, a myotubularin phosphatase family member, functions in the control of PI(3)P-dependent signaling and the maintenance of endosomal system integrity. In addition, this work defined an essential overlapping role for lipid phosphatases in the regulation of 3' phosphoinositides in yeast.

Aminopeptidases↗

PubMed2004

Regulation of epidermal growth factor receptor degradation by heterotrimeric Galphas protein.

Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Galphas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Galphas expression by RNA interference (RNAi) delays receptor degradation. We also show that Galphas and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Galphas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Galphas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Galphas on endosomes. Furthermore, knock-down of both Hrs and Galphas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Galphas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Galphas in EGF receptor degradation and provide mechanistic insights into the function of Galphas in endocytic sorting.

Animals↗

PubMed1997

Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.

The epithelial Na+ channel (ENaC), composed of three subunits (alpha beta gamma), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2 approximately 1 h) that is ubiquitinated in vivo on the alpha and gamma (but not beta) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of gamma ENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in alpha ENaC, but not in beta ENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled alpha beta gamma ENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels.

Acetylcysteine↗

PubMed2001

Hrs recruits clathrin to early endosomes.

The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.

Amino Acid Sequence↗

PubMed2000

The neurofibromatosis 2 tumor suppressor protein interacts with hepatocyte growth factor-regulated tyrosine kinase substrate.

The neurofibromatosis 2 tumor suppressor protein schwannomin/merlin is commonly mutated in schwannomas and meningiomas. Schwannomin, a member of the 4.1 family of proteins, which are known to link the cytoskeleton to the plasma membrane, has little known function other than its ability to suppress tumor growth. Using yeast two-hybrid interaction cloning, we identified the HGF-regulated tyrosine kinase substrate (HRS) as a schwannomin interactor. We verified the interaction by both immunoprecipitation of endogenous HRS with endogenous schwannomin in vivo as well as by using bacterially purified HRS and schwannomin in vitro. We narrowed the regions of interaction to include schwannomin residues 256-579 and HRS residues from 480 to the end of either of two HRS isoforms. Schwannomin molecules with a L46R, L360P, L535P or Q538P missense mutation demonstrated reduced affinity for HRS binding. As HRS is associated with early endosomes and may mediate receptor translocation to the lysosome, we demonstrated that schwannomin and HRS co-localize at endosomes using the early endosome antigen 1 in STS26T Schwann cells by indirect immunofluorescence. The identification of schwannomin as a HRS interactor implicates schwannomin in HRS-mediated cell signaling.

Adult↗

PubMed2004

Association with Hrs is required for the early endosomal localization, stability, and function of STAM.

Members of the STAM family of proteins, STAM1 and STAM2, are associated with Hrs through their coiled-coil regions. Both Hrs and STAM bind ubiquitin and are involved in endosomal sorting of ubiquitinated cargo proteins for trafficking to the lysosome. Here we examined the biological significance of STAM binding to Hrs. Endogenous STAM1 and STAM2 were mostly localized on the early endosome, suggesting that they are resident endosomal proteins. A STAM2 mutant that lacks the coiled-coil region and does not bind Hrs, in contrast, mislocalized to the cytoplasm. Deletion of a region located N-terminal to the coiled-coil region and conserved among STAM proteins also severely affected Hrs binding and the endosomal localization of STAM2, suggesting that this region is also involved in these activities. Depletion of endogenous Hrs by RNA interference similarly caused the mislocalization of exogenously expressed STAM2 to the cytoplasm. These results indicate that STAM is localized to the early endosome by binding to Hrs on the target membrane. In addition, the expression level of endogenous STAM proteins was drastically reduced in Hrs-depleted cells, suggesting that STAM is stabilized by binding to Hrs. Finally, STAM2 mutants lacking the Hrs-binding activity were defective in causing the enlargement of early endosomes, accumulating ubiquitinated proteins on this aberrant organelle, and inhibiting the degradation of ligand-activated epidermal growth factor receptors, suggesting that the association with Hrs is a prerequisite for STAM function.

Adaptor Proteins, Signal Transducing↗

PubMed2005

ESCRT-I components of the endocytic machinery are required for Rim101-dependent ambient pH regulation in the yeast Yarrowia lipolytica.

Ambient pH signalling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Insertional mutagenesis in the yeast Yarrowia lipolytica identified two components of the endosome-associated ESCRT-I complex involved in multivesicular body (MVB) vesicle formation, YlVps28p and YlVps23p. They were shown to be required at alkaline pH, like Rim factors, for transcriptional activation of alkaline-induced genes and repression of acid-induced genes. The constitutively active YlRIM101-1119 allele, which suppresses the pH-signalling defects of Ylrim mutations, also suppresses Ylvps defects in pH response, but not in endocytosis. The contribution of the ESCRT-III component Snf7p could not be assessed due to the essential nature of this component in Y. lipolytica. Unlike Rim factors, YlVps4p, a component of the MVB pathway acting downstream from ESCRT complexes, seems not to be required for the alkaline response. In Y. lipolytica, all vps mutations including those affecting YlVPS4, affected growth at acidic pH, a feature not exhibited by Ylrim mutations. These results suggest that Rim and Vps pathways cooperate in ambient pH signalling and that this relation is conserved across the full range of hemiascomycetous yeasts.

DNA-Binding Proteins↗

PubMed2006

Clathrin anchors deubiquitinating enzymes, AMSH and AMSH-like protein, on early endosomes.

Endosomal sorting of ubiquitinated membrane proteins for trafficking to lysosomes is executed by a complex of two ubiquitin-binding proteins, Hrs and STAM, that localizes on a microdomain of early endosomes with a flat clathrin coat. AMSH is a deubiquitinating enzyme that interacts with STAM and is implicated in the down-regulation of epidermal growth factor receptor. AMSH has a close homolog, AMSH-like protein (AMSH-LP). Here we show that AMSH-LP is also a deubiquitinating enzyme that acts on early endosomes. We further show that AMSH and AMSH-LP bind to the terminal domain of clathrin heavy chain via a novel clathrin-binding site conserved between these proteins. Exogenously expressed AMSH and AMSH-LP co-localized with clathrin on early endosomes. However, deletion of the clathrin-binding site from the proteins, as well as RNA interference-mediated depletion of clathrin heavy chain, resulted in a failure of AMSH and AMSH-LP to localize on endosomes. In contrast, a mutant of AMSH that lacks the ability to bind STAM localized normally on endosomes. We suggest that AMSH and AMSH-LP are anchored on the early endosomal membrane via interaction with the clathrin coat.

Amino Acid Sequence↗

PubMed2004

Role of LBPA and Alix in multivesicular liposome formation and endosome organization.

What are the components that control the assembly of subcellular organelles in eukaryotic cells? Although membranes can clearly be distorted by cytosolic factors, very little is known about the intrinsic mechanisms that control the biogenesis, shape, and organization of organellar membranes. Here, we found that the unconventional phospholipid lysobisphosphatidic acid (LBPA) could induce the formation of multivesicular liposomes that resembled the multivesicular endosomes that exist where this lipid is found in vivo. This process depended on the same pH gradient that exists across endosome membranes in vivo and was selectively controlled by Alix. In turn, Alix regulated the organization of LBPA-containing endosomes in vivo.

Animals↗

PubMed2005

Deletions of endocytic components VPS28 and VPS32 affect growth at alkaline pH and virulence through both RIM101-dependent and RIM101-independent pathways in Candida albicans.

Ambient pH signaling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Recent evidences in the fungi Aspergillus nidulans, Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida albicans suggested that components of endosomal sorting complexes required for transport (ESCRT) involved in endocytic trafficking were needed for signal transduction along the Rim pathway. In this study, we confirm these findings with C. albicans and show that Vps28p (ESCRT-I) and Vps32p/Snf7p (ESCRT-III) are required for the transcriptional regulation of known targets of the Rim pathway, such as the PHR1 and PHR2 genes encoding cell surface proteins, which are expressed at alkaline and acidic pH, respectively. We additionally show that deletion of these two VPS genes, particularly VPS32, has a more drastic effect than a RIM101 deletion on growth at alkaline pH and that this effect is only partially suppressed by expression of a constitutively active form of Rim101p. Finally, in an in vivo mouse model, both vps null mutants were significantly less virulent than a rim101 mutant, suggesting that VPS28 and VPS32 gene products affect virulence both through Rim-dependent and Rim-independent pathways.

Alkalies↗

PubMed2003

Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression.

Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5') suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release. We observed that a mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release. We also determined the effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. We propose that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting.

Amino Acid Motifs↗

PubMed2000

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.

Amino Acid Sequence↗