Effects of human a3 and a4 mutations that result in osteopetrosis and distal renal tubular acidosis on yeast V-ATPase expression and activity.

摘要

V-ATPases are multimeric proton pumps. The 'a' subunit is encoded by four isoforms (a1-a4) in mammals and two (Vph1p and Stv1p) in yeast. 'a3' is enriched in osteoclasts and is essential for bone resorption while a4 is expressed in the distal nephron and acidifies urine. Mutations to human a3 and a4 genes result in osteopetrosis and distal renal tubular acidosis, respectively. We have recreated human a3 (G405R, R444L) and a4 (P524L, G820R) mutations in conserved regions of the yeast V-ATPase 'a' subunit ortholog, Vph1p as: a3 (G424R, R462L), a4 (W520L, G812R). 'a3' (G424R, R462L) mutations had near wild-type activity and wild-type expression of V-ATPase subunits. 'a4' mutation G812R had severely reduced activity and wild-type expression. 'a4' mutation W520L was dominant negative in yeast, as overexpression of wild-type yeast 'a' isoforms Vph1p or Stv1p did not restore activity or expression. Deletion of endoplasmic reticulum assembly factors partially rescued this phenotype.