C-Terminal truncations of the Saccharomyces cerevisiae PMA1 H+-ATPase have major impacts on protein conformation, trafficking, quality control, and function

Abstract

The C-terminal tail of yeast plasma membrane (PM) H+-ATPase extends approximately 38 amino acids beyond the final membrane- spanning segment (TM10) of the protein and is known to be required for successful trafficking, stability, and regulation of enzyme activity. To carry out a detailed functional survey of the entire length of the tail, we generated 15 stepwise truncation mutants. Eleven of them, lacking up to 30 amino acids from the extreme terminus, were able to support cell growth, even though there were detectable changes in plasma membrane expression, protein stability, and ATPase activity. Three functionally distinct regions of the C terminus could be defined. (i) Truncations upstream of Lys889, removing more than 30 amino acid residues, yielded no viable mutants, and conditional expression of such constructs supported the conclusion that the stretch from Ala881 (at the end of TM10) to Gly888 is required for stable folding and PM targeting. (ii) The stretch between Lys889 and Lys916, a region known to be subject to kinase-mediated posttranslational modification, was shown here to be ubiquitinated in carbon-starved cells as part of cellular quality control and to be essential for normal ATPase folding and stability, as well as for autoinhibition of ATPase activity during glucose starvation. (iii) Finally, removal of even one or two residues (Glu917 and Thr918) from the extreme C terminus led to visibly reduced expression of the ATPase at the plasma membrane. Thus, the C terminus is much more than a simple appendage and profoundly influences the structure, biogenesis, and function of the yeast H+-ATPase. © 2014, American Society for Microbiology. All Rights Reserved.