The human papillomavirus type 16 E5 protein alters vacuolar H+-ATPase function and stability in Saccharomyces cerevisiae

The human papillomavirus 16 (HPV-16) E5 oncoprotein is a small integral membrane protein that binds to the 16-kDa subunit of the vacuolar H+-ATPase (v-ATPase). Conservation within the family of v-ATPases prompted us to look to Saccharomyces cerevisiae as a potential model organism for E5 study. The E5 open reading frame, driven by a galactose-inducible promoter, was integrated into the yeast genome, and the resulting strain demonstrated a nearly complete growth arrest at neutral pH, consistent with defects associated with yeast: v-ATPase mutants. Furthermore, this strain demonstrated a severe reduction in pH-dependent and v-ATPase-dependent vacuolar localization of fluorescent markers. Overexpression of the yeast 16-kDa subunit homolog partially suppressed E5-associated growth defects. E5 expression was correlated with a disassociation of the integral (V-o) and peripheral (V-i) v-ATPase sub-complexes, as well as a dramatic reduction of the steady-state levels of one mature V-o subunit and the concomitant accumulation of its major proteolytic fragment, with unchanged levels of two V-i subunits. Similar analyses of selected E5 mutants in yeast demonstrated a correlation between E5 biology and v-ATPase disruption. Our observations suggest that wild-type HPV-16 E5 acts during the assembly of the v-ATPase to inhibit, either directly or indirectly, V-o stability and complex formation. (C) 2001 Academic Press.