The Spf1p P5A-ATPase “arm-like” domain is not essential for ATP hydrolysis but its deletion impairs autophosphorylation

GRENON, PAULA; CORRADI, GERARDO R.; PETROVICH, GUIDO D.; MAZZITELLI, LUCIANA R.; ADAMO, HUGO P.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2021 vol. 563 p. 113 - 118

0006-291X

The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the ?arm-like? domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the ?arm-like? domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the ?arm-like? domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.