The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa
Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit “a” has four isoforms, a1–a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KX<sub>n</sub>K(R)IK(R), conserve...
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MDPI AG
2024-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/4/2170 |
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| author | Anh Chu Yeqi Yao Miroslawa Glibowicka Charles M. Deber Morris F. Manolson |
| author_facet | Anh Chu Yeqi Yao Miroslawa Glibowicka Charles M. Deber Morris F. Manolson |
| author_sort | Anh Chu |
| collection | DOAJ |
| description | Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit “a” has four isoforms, a1–a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KX<sub>n</sub>K(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa. |
| first_indexed | 2024-03-07T22:28:30Z |
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| language | English |
| last_indexed | 2024-03-07T22:28:30Z |
| publishDate | 2024-02-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-4d7705eff4974f0299322ffff43f505d2024-02-23T15:20:03ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-02-01254217010.3390/ijms25042170The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis LaxaAnh Chu0Yeqi Yao1Miroslawa Glibowicka2Charles M. Deber3Morris F. Manolson4Faculty of Dentistry, University of Toronto, Toronto M5G 1G6, ON, CanadaFaculty of Dentistry, University of Toronto, Toronto M5G 1G6, ON, CanadaDivision of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, ON, CanadaDivision of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, ON, CanadaFaculty of Dentistry, University of Toronto, Toronto M5G 1G6, ON, CanadaVacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit “a” has four isoforms, a1–a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KX<sub>n</sub>K(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa.https://www.mdpi.com/1422-0067/25/4/2170V-ATPasesV-ATPase a2 isoformsprotein–lipid interactionphosphoinositidesPI(4)Pcutis laxa |
| spellingShingle | Anh Chu Yeqi Yao Miroslawa Glibowicka Charles M. Deber Morris F. Manolson The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa International Journal of Molecular Sciences V-ATPases V-ATPase a2 isoforms protein–lipid interaction phosphoinositides PI(4)P cutis laxa |
| title | The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa |
| title_full | The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa |
| title_fullStr | The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa |
| title_full_unstemmed | The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa |
| title_short | The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa |
| title_sort | human mutation k237 v238del in a putative lipid binding motif within the v atpase a2 isoform suggests a molecular mechanism underlying cutis laxa |
| topic | V-ATPases V-ATPase a2 isoforms protein–lipid interaction phosphoinositides PI(4)P cutis laxa |
| url | https://www.mdpi.com/1422-0067/25/4/2170 |
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