ATP6V1H encodes V-type proton ATPase subunit H (483 amino acids, ~57 kDa), the regulatory subunit H of the V1 peripheral domain of the vacuolar-type H+-ATPase (V-ATPase). Subunit H is present as a single copy in the V1 complex and has a dual regulatory role: in the assembled V-ATPase holoenzyme it supports proton pump activity, while in the free cytosolic V1 complex (dissociated from V0 during regulated disassembly) it inhibits futile ATP hydrolysis. Beyond its structural role in the V-ATPase, subunit H directly binds to AP2M1 (the medium chain mu2 of adaptor protein complex 2) through armadillo repeat domains spanning residues 133-363, physically connecting the V-ATPase to the clathrin-mediated endocytic machinery. The protein was originally identified as Nef-binding protein 1 (NBP1) and is the human ortholog of yeast Vma13p. HIV-1 and SIV Nef exploit the subunit H-AP2M1 interaction to forcibly internalize CD4 from infected cell surfaces, but this reflects co-option of a normal cellular endocytic function. Two isoforms exist (Q9UI12-1 and Q9UI12-2); isoform 2 differs at residues 176-193. The protein localizes to lysosomal and endosomal membranes (as part of assembled V-ATPase), to the cytosol (as part of free V1 complex), and at clathrin-coated vesicle membranes. ATP6V1H is ubiquitously expressed.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000221 vacuolar proton-transporting V-type ATPase, V1 domain | IBA GO_REF:0000033 | ACCEPT | Summary: Subunit H is a genuine V1 domain component confirmed by cryo-EM and biochemical characterization. Reason: V1 domain membership is well established. Subunit H is a regulatory component of the V1 complex, one copy per holoenzyme (PMID:33065002, PMID:9442887). |
| GO:0007042 lysosomal lumen acidification | IBA GO_REF:0000033 | ACCEPT | Summary: IBA phylogenetic transfer; lysosomal acidification is the core biological output of the assembled V-ATPase containing subunit H. Reason: Lysosomal lumen acidification is the primary downstream consequence of V-ATPase proton pumping. As a regulatory subunit essential for V-ATPase activity, H is rightly annotated as involved in this process. |
| GO:0097401 synaptic vesicle lumen acidification | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA transfer for synaptic vesicle lumen acidification; non-core neuronal context for this ubiquitously expressed subunit. Reason: Neuronal synaptic vesicle acidification is a non-core context for this ubiquitous regulatory subunit. |
| GO:0000221 vacuolar proton-transporting V-type ATPase, V1 domain | IEA GO_REF:0000002 | ACCEPT | Summary: IEA from InterPro; V1 domain membership is experimentally established. Reason: V1 domain membership is supported by structural and functional data. |
| GO:0016020 membrane | IEA GO_REF:0000117 | MODIFY | Summary: IEA ARBA for membrane localization; overly general term but consistent with lysosomal/endosomal/plasma membrane localization. Reason: Generic membrane is too imprecise. The more specific terms lysosomal membrane (HDA), endosome membrane (NAS), and plasma membrane (NAS) already exist in the annotation set. The IDA annotation to GO:0016020 from PMID:33065002 directly contextualizes which membrane is meant. Proposed replacements: lysosomal membrane |
| GO:0030665 clathrin-coated vesicle membrane | IEA GO_REF:0000044 | ACCEPT | Summary: IEA from UniProt subcellular location mapping; consistent with the documented AP-2 (clathrin adaptor) interaction of subunit H. Reason: Subunit H directly binds AP2M1 (PMID:12032142), which is a component of clathrin-coated vesicle machinery. Localization at clathrin-coated vesicle membrane is consistent with this interaction. Supporting Evidence: PMID:12032142 V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo |
| GO:0046961 proton-transporting ATPase activity, rotational mechanism | IEA GO_REF:0000002 | ACCEPT | Summary: IEA from InterPro; proton-transporting ATPase rotational mechanism is the molecular activity of the V-ATPase complex. Reason: The V-ATPase uses a rotational mechanism for proton translocation. Subunit H contributes to this complex activity as a regulatory component. |
| GO:1902600 proton transmembrane transport | IEA GO_REF:0000002 | ACCEPT | Summary: IEA from InterPro; proton transmembrane transport is the core biological process of the V-ATPase. Reason: Proton transmembrane transport is the fundamental function of the V-ATPase complex. Subunit H is essential for this activity as a regulatory component. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Generic protein binding from binary interactome reference map; uninformative over-annotation. Reason: High-throughput interactome dataset. The specific informative interaction is with AP2M1 (PMID:12032142), not the generic protein binding term. |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | MARK AS OVER ANNOTATED | Summary: Generic protein binding from neurodegenerative disease interactome; uninformative over-annotation. Reason: High-throughput interactome dataset; protein binding does not describe specific molecular function of subunit H. |
| GO:0098850 extrinsic component of synaptic vesicle membrane | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: IEA Ensembl Compara transfer; non-core neuronal context for this ubiquitous subunit. Reason: Synaptic vesicle context is non-core for this ubiquitously expressed regulatory subunit. |
| GO:0005829 cytosol | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: IDA from immunofluorescence curation; cytosolic localization reflects the free V1 complex state. Reason: Cytosolic localization is a genuine functional state for subunit H. The free V1 complex is present in the cytosol when dissociated from V0, and subunit H specifically inhibits futile ATP hydrolysis in this context. |
| GO:0000139 Golgi membrane | NAS PMID:32001091 Structure and Roles of V-type ATPases. | KEEP AS NON CORE | Summary: NAS from V-ATPase review; Golgi membrane localization is mentioned for V-ATPase generally. Not specific to subunit H but consistent with the review's description of V-ATPase distribution. Reason: Golgi membrane localization is supported only by NAS from a general V-ATPase review. While V-ATPase does localize to Golgi, this is not a core context for the H subunit. |
| GO:0005765 lysosomal membrane | NAS PMID:32001091 Structure and Roles of V-type ATPases. | ACCEPT | Summary: NAS from V-ATPase review; lysosomal membrane localization is the core localization for the assembled V-ATPase holoenzyme. Reason: Lysosomal membrane localization is well supported and is the primary localization of the assembled V-ATPase. Also supported by HDA mass spectrometry (PMID:17897319). |
| GO:0005886 plasma membrane | NAS PMID:32001091 Structure and Roles of V-type ATPases. | KEEP AS NON CORE | Summary: NAS from V-ATPase review; plasma membrane V-ATPase in specialized cell types (e.g., kidney intercalated cells). Reason: Plasma membrane localization is a non-core context for this ubiquitous subunit; it occurs in specialized cells. The more relevant localization is lysosomal/endosomal. |
| GO:0007035 vacuolar acidification | NAS PMID:32001091 Structure and Roles of V-type ATPases. | ACCEPT | Summary: NAS from V-ATPase review; vacuolar acidification is the core biological process downstream of V-ATPase proton pumping. Reason: Vacuolar acidification is the primary biological function of V-ATPase activity. Subunit H as a regulatory component of the V-ATPase is appropriately annotated to this process. |
| GO:0007042 lysosomal lumen acidification | NAS PMID:32001091 Structure and Roles of V-type ATPases. | ACCEPT | Summary: NAS from V-ATPase review; lysosomal lumen acidification is the core functional output of lysosome-localized V-ATPase. Reason: Lysosomal lumen acidification is the primary biological process driven by the V-ATPase at the lysosomal membrane. Subunit H is a required regulatory component of this activity. |
| GO:0007042 lysosomal lumen acidification | NAS PMID:33065002 Structures of a Complete Human V-ATPase Reveal Mechanisms of... | ACCEPT | Summary: NAS from cryo-EM structure paper; lysosomal lumen acidification is the core function of the V-ATPase complex. Reason: The structure paper describes V-ATPase function in intracellular acidification. Lysosomal lumen acidification is a core function. |
| GO:0010008 endosome membrane | NAS PMID:32001091 Structure and Roles of V-type ATPases. | ACCEPT | Summary: NAS from V-ATPase review; endosome membrane localization of assembled V-ATPase is well established. Reason: Endosomal membrane localization of V-ATPase is well established and important for receptor-mediated endocytosis and iron release from transferrin. |
| GO:0016020 membrane | IDA PMID:33065002 Structures of a Complete Human V-ATPase Reveal Mechanisms of... | MODIFY | Summary: IDA from cryo-EM structure study; this directly shows subunit H as part of the membrane-associated V-ATPase holoenzyme. Reason: The cryo-EM structure places subunit H in the V-ATPase complex at membranes. The generic membrane term is less informative than lysosomal membrane. Suggest retaining but noting more specific terms are preferred. Proposed replacements: lysosomal membrane |
| GO:0033176 proton-transporting V-type ATPase complex | NAS PMID:33065002 Structures of a Complete Human V-ATPase Reveal Mechanisms of... | ACCEPT | Summary: NAS from cryo-EM structure paper; V-ATPase complex membership is well established. Reason: Subunit H is a confirmed component of the V-ATPase holoenzyme complex. |
| GO:0048388 endosomal lumen acidification | NAS PMID:32001091 Structure and Roles of V-type ATPases. | ACCEPT | Summary: NAS from V-ATPase review; endosomal lumen acidification is a core biological process downstream of V-ATPase activity at endosomes. Reason: Endosomal acidification is required for receptor-mediated endocytosis completion and nutrient release. V-ATPase is the primary driver; subunit H is a required component. |
| GO:0051452 intracellular pH reduction | NAS PMID:32001091 Structure and Roles of V-type ATPases. | KEEP AS NON CORE | Summary: NAS from V-ATPase review; intracellular pH reduction is a broader term encompassing all V-ATPase-dependent compartment acidification. Reason: Intracellular pH reduction is a general consequence of V-ATPase activity. More specific annotations to lysosomal and endosomal lumen acidification are already present and are preferable. This broader term is non-core. |
| GO:0061795 Golgi lumen acidification | NAS PMID:32001091 Structure and Roles of V-type ATPases. | KEEP AS NON CORE | Summary: NAS from V-ATPase review; Golgi lumen acidification is a downstream consequence of V-ATPase activity at Golgi membranes. Reason: Golgi acidification is a non-core downstream function. Lysosomal and endosomal acidification are the primary core contexts. |
| GO:1902600 proton transmembrane transport | NAS PMID:33065002 Structures of a Complete Human V-ATPase Reveal Mechanisms of... | ACCEPT | Summary: NAS from cryo-EM structure paper; proton transmembrane transport is the core molecular function of the V-ATPase complex. Reason: Proton transmembrane transport is the fundamental process of the V-ATPase. Subunit H is essential for this activity. |
| GO:0000221 vacuolar proton-transporting V-type ATPase, V1 domain | ISS GO_REF:0000024 | ACCEPT | Summary: ISS manual ortholog transfer; V1 domain membership is experimentally established. Reason: ISS consistent with direct experimental evidence for V1 domain membership. |
| GO:0005515 protein binding | IPI PMID:25659576 TM9SF4 is a novel V-ATPase-interacting protein that modulate... | MARK AS OVER ANNOTATED | Summary: IPI from TM9SF4/V-ATPase interaction study in colon cancer; TM9SF4 co-immunoprecipitates with ATP6V1H. This is a specific interaction study but the GO:0005515 annotation is still uninformative. Reason: While TM9SF4 interaction with ATP6V1H is documented (PMID:25659576), the protein binding annotation is uninformative. The interaction is in a cancer cell context and the normal physiological relevance is unclear. |
| GO:0016241 regulation of macroautophagy | NAS PMID:22982048 Lipofuscin is formed independently of macroautophagy and lys... | MARK AS OVER ANNOTATED | Summary: NAS annotation; the cited paper uses V-ATPase disruption as a tool to impair lysosomal activity. Does not specifically implicate subunit H in macroautophagy regulation. Reason: The cited study does not demonstrate that ATP6V1H specifically regulates macroautophagy; it uses generic V-ATPase disruption to block lysosomal function. This is an over-annotation of a generic downstream consequence of V-ATPase disruption. |
| GO:0070062 extracellular exosome | HDA PMID:19199708 Proteomic analysis of human parotid gland exosomes by multid... | MARK AS OVER ANNOTATED | Summary: HDA from parotid gland exosome proteomics; likely a contaminant in exosome fractions. Reason: Extracellular exosome identification in proteomics is likely contamination. Not a primary localization for a V1 peripheral complex subunit. |
| GO:0070062 extracellular exosome | HDA PMID:19056867 Large-scale proteomics and phosphoproteomics of urinary exos... | MARK AS OVER ANNOTATED | Summary: HDA from urinary exosome proteomics; likely a contaminant in exosome fractions. Reason: Extracellular exosome identification in proteomics is likely contamination. Not a primary localization for a V1 subunit. |
| GO:0005765 lysosomal membrane | HDA PMID:17897319 Integral and associated lysosomal membrane proteins. | ACCEPT | Summary: HDA from lysosomal membrane proteomics; directly supports lysosomal membrane localization as part of the assembled V-ATPase. Reason: Mass spectrometry in lysosome-enriched fractions directly identifies subunit H at the lysosomal membrane. Supporting Evidence: PMID:17897319 Integral and associated lysosomal membrane proteins |
| GO:0005829 cytosol | TAS Reactome:R-HSA-1222516 | KEEP AS NON CORE | Summary: Reactome TAS annotation; cytosolic localization reflects free V1 complex or V1H in its adaptor role during endocytic events. Reason: Cytosolic localization is well-established for the free V1 complex. Subunit H has a specific regulatory role in the cytosolic free V1 state (inhibiting futile ATP hydrolysis). Multiple independent sources confirm this. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-167597 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol in Nef/CD4 endocytosis context; V1H bridges cytosolic Nef to the AP-2 endocytic complex. Reason: In the Nef endocytosis pathway, V1H functions as a cytosolic adaptor. This is consistent with the documented AP2M1 binding. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-167601 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol in CD4 degradation pathway; consistent. Reason: Cytosolic localization context; consistent with subunit H adaptor function. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-182171 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol in CD8 degradation pathway; consistent. Reason: Cytosolic localization context; consistent with subunit H adaptor function. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-182198 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol in CD8 internalization; consistent. Reason: Cytosolic localization context; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-5252133 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol; free V1 complex context. Reason: Cytosolic localization of free V1 complex; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-74723 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol; free V1 complex context. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-917841 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol; free V1 complex context. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9636397 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol; Mycobacterium PtpA binds ATP6V1H context. Reason: Cytosolic localization; consistent with free V1 complex. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9639286 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol in mTORC1 signaling context. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9640167 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9640168 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9640175 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9640195 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9645598 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9645608 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9646468 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9858916 | KEEP AS NON CORE | Summary: Reactome TAS annotation for cytosol. Reason: Cytosolic localization; consistent. |
| GO:0005886 plasma membrane | TAS Reactome:R-HSA-167537 | KEEP AS NON CORE | Summary: Reactome TAS annotation for plasma membrane in Nef/CD4 complex context; V1H at plasma membrane bridges Nef to AP-2 for CD4 internalization. Reason: Plasma membrane localization in the Nef/CD4 endocytosis context reflects the adaptor function but is non-core relative to lysosomal/endosomal localization. |
| GO:0005886 plasma membrane | TAS Reactome:R-HSA-167597 | KEEP AS NON CORE | Summary: Reactome TAS for plasma membrane in CD4 internalization context; non-core. Reason: Non-core context; same reasoning as above. |
| GO:0005886 plasma membrane | TAS Reactome:R-HSA-182186 | KEEP AS NON CORE | Summary: Reactome TAS for plasma membrane in CD8/Nef complex context; non-core. Reason: Non-core context; plasma membrane in Nef pathway. |
| GO:0005886 plasma membrane | TAS Reactome:R-HSA-182198 | KEEP AS NON CORE | Summary: Reactome TAS for plasma membrane in CD8 internalization context; non-core. Reason: Non-core context. |
| GO:0005515 protein binding | IPI PMID:11179428 Negative factor from SIV binds to the catalytic subunit of t... | MARK AS OVER ANNOTATED | Summary: IPI from SIV Nef/V-ATPase study; the specific interaction is SIV Nef with subunit H, exploiting the normal AP-2 adaptor function. The protein binding annotation is uninformative. Reason: The SIV Nef-H interaction is documented (PMID:11179428) but the generic protein binding term does not capture the biology. The relevant specific function is the AP-2 medium chain (AP2M1) binding. |
| GO:0000221 vacuolar proton-transporting V-type ATPase, V1 domain | NAS PMID:9442887 Structure, function and regulation of the vacuolar (H+)-ATPa... | ACCEPT | Summary: NAS from Stevens and Forgac review; V1 domain membership is well-established. Reason: The 1997 Stevens and Forgac review is the foundational reference for V-ATPase V1 subunit composition including subunit H. Supporting Evidence: PMID:9442887 The peripheral V1 domain, a 500-kDa complex responsible for ATP hydrolysis, contains at least eight different subunits of molecular weight 70-13 (subunits A-H) |
| GO:0005515 protein binding | IPI PMID:12032142 Subunit H of the V-ATPase binds to the medium chain of adapt... | MARK AS OVER ANNOTATED | Summary: IPI from Geyer et al. 2002; this study demonstrated specific interaction with AP2M1. The protein binding annotation is uninformative but the underlying interaction is important. Reason: The specific interaction with AP2M1 (mu2 adaptin) is meaningful and well-documented, but GO:0005515 protein binding is uninformative. A more specific annotation to AP-2 adaptor binding or clathrin adaptor binding would be more informative. |
| GO:0005515 protein binding | IPI PMID:9620685 Interactions between HIV1 Nef and vacuolar ATPase facilitate... | MARK AS OVER ANNOTATED | Summary: IPI from Lu et al. 1998; interaction with HIV-1 Nef documented. Generic protein binding is uninformative. Reason: Generic protein binding annotation; the specific interaction is with HIV-1 Nef (a pathogen protein) and does not reflect normal cellular function. |
| GO:0006897 endocytosis | IDA PMID:12032142 Subunit H of the V-ATPase binds to the medium chain of adapt... | ACCEPT | Summary: IDA experimental evidence that V1H contributes to endocytosis via AP-2 interaction; this is a legitimate specific function of subunit H. Reason: Geyer et al. 2002 demonstrated that V1H connects to the endocytic machinery through AP2M1 interaction, and V1H-Nef chimeras can drive CD4 internalization. This is genuine experimental evidence for H subunit involvement in clathrin-mediated endocytosis. Supporting Evidence: PMID:12032142 V1H can function as an adaptor for interactions between Nef and AP-2 |
| GO:0007035 vacuolar acidification | NAS PMID:9442887 Structure, function and regulation of the vacuolar (H+)-ATPa... | ACCEPT | Summary: NAS from foundational V-ATPase review; vacuolar acidification is the core biological process. Reason: Vacuolar acidification is a core biological process driven by V-ATPase. The Stevens and Forgac review is a valid reference for this NAS annotation. Supporting Evidence: PMID:9442887 The vacuolar (H+)-ATPases (or V-ATPases) function in the acidification of intracellular compartments in eukaryotic cells |
| GO:0016887 ATP hydrolysis activity | NAS PMID:9442887 Structure, function and regulation of the vacuolar (H+)-ATPa... | ACCEPT | Summary: NAS from foundational V-ATPase review; subunit H contributes to ATP hydrolysis activity of the V-ATPase complex. Reason: ATP hydrolysis is the biochemical activity of the V1 domain. Subunit H is a regulatory component that modulates this activity. The contributes_to qualifier is appropriate. Supporting Evidence: PMID:9442887 The peripheral V1 domain, a 500-kDa complex responsible for ATP hydrolysis, contains at least eight different subunits of molecular weight 70-13 (subunits A-H) |
| GO:0030234 enzyme regulator activity | NAS PMID:9442887 Structure, function and regulation of the vacuolar (H+)-ATPa... | ACCEPT | Summary: NAS for enzyme regulator activity; subunit H is the regulatory H subunit that modulates V-ATPase ATP hydrolysis in assembled versus free V1 states. Reason: Subunit H has a documented regulatory function — it inhibits futile ATP hydrolysis in the free V1 complex and activates the pump when assembled with V0. This enzyme regulator activity is a genuinely specific function of the H subunit distinguishing it from other V1 subunits. |
| GO:1902600 proton transmembrane transport | NAS PMID:9442887 Structure, function and regulation of the vacuolar (H+)-ATPa... | ACCEPT | Summary: NAS from foundational V-ATPase review; proton transmembrane transport is the core function. Reason: Proton transmembrane transport is the core function of the V-ATPase. Subunit H as a regulatory component is appropriately annotated to this process. |
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Download this section (compressed HTML)Q: Does the AP2M1-binding function of subunit H reflect a conserved role of V-ATPase in clathrin-coated vesicle biogenesis, or is the H subunit unusually specialized for this endocytic adaptor role compared to other V1 subunits?
Suggested experts: Geyer M, Peterlin BM
Q: How does the regulatory switch of subunit H work mechanistically — what structural changes occur in H between the free V1 state (ATP hydrolysis inhibited) and the assembled holoenzyme state (ATP hydrolysis activated)?
Suggested experts: Forgac M, Rubinstein JL
Experiment: Generate separation-of-function mutations in ATP6V1H that disrupt AP2M1 binding (within residues 133-363) without affecting V1 complex assembly or proton pump activity. Assess clathrin-mediated endocytosis of physiological cargo (transferrin receptor, EGF receptor) in cells expressing mutant versus wild-type H subunit.
Hypothesis: The AP2M1-binding function of subunit H is required for normal clathrin-mediated endocytosis independent of V-ATPase proton pumping.
Type: structure-function mutagenesis and receptor internalization assay
Experiment: Quantify the ratio of V1H in membrane-bound (V-ATPase assembled) versus cytosolic (free V1) fractions under nutrient replete and starved conditions by subcellular fractionation and quantitative proteomics, and measure V1-ATPase activity in each fraction to directly test the inhibitory role of H in the free state.
Hypothesis: Regulated V1/V0 disassembly differentially affects the H subunit regulatory function.
Type: subcellular fractionation and ATPase activity assay
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