Dmxl1 Is an Essential Mammalian Gene that Is Required for V-ATPase Assembly and Function In Vivo

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The vacuolar-type H+-ATPase (V-ATPase) is essential for cellular acidification and its dysfunction is linked to various diseases.
• Proper assembly of V-ATPase's V1 and VO domains into a functional holoenzyme is critical for its proton-pumping activity.
• The molecular mechanisms regulating V-ATPase assembly in mammals remain largely uncharacterized.

Purpose of the Study:

• To investigate the role of Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), also known as Rabconnectin-3A, as a potential V-ATPase assembly factor in mammals.
• To determine the impact of Dmxl1 deficiency on V-ATPase function and assembly in kidney intercalated cells (ICs).

Main Methods:

• Generation of kidney intercalated cell-specific Dmxl1 knockout (KO) mice.
• Analysis of urine pH, V-ATPase subunit expression and localization via Western blotting and immunofluorescence.
• Subcellular fractionation to assess V1 domain association with the membrane.
• Proximity ligation assay to evaluate V1 and VO subunit association.

Main Results:

• Dmlxl1 KO mice exhibited high urine pH, indicative of impaired V-ATPase function in kidney ICs.
• Loss of Dmxl1 led to decreased expression of the V-ATPase B1 subunit and reduced colocalization of V1 and VO subunits.
• Subcellular fractionation revealed diminished V1 domain association with the membrane fraction in Dmxl1-deficient cells.
• Proximity ligation assays confirmed reduced association between V-ATPase B1 and a4 subunits in Dmxl1 KO ICs.

Conclusions:

• Dmxl1 is a bona fide mammalian V-ATPase assembly factor.
• Loss of Dmxl1 disrupts V-ATPase holoenzyme assembly, leading to impaired proton pumping activity.
• Dmxl1 likely facilitates the recruitment of the V1 domain to the membrane for proper assembly with the VO domain.