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Abstract
Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.
Highlights
With more subtle abnormalities in folding, are transported further along the secretory pathway to reach the Golgi or even the plasma membrane, before being recognized as abnormal and committed to the endosomal/vacuolar pathway for degradation. In addition to their use in mapping amino acid residues within the Pma1 polypeptide that are important for folding and trafficking, both kinds of mutant have been used to identify novel components of the yeast secretory pathway, such as Eps1p, a protein-disulfide isomerase implicated in ER quality control [7], and Ast1p, a protein involved in transport from the Golgi to the plasma membrane [9]
To dissect the role of the C-terminal portion of yeast Pma1 ATPase in biogenesis and trafficking, we have constructed a set of mutants with truncations after each of the last six transmembrane segments, from M5 through M10
A series of truncations have been used to examine the role of the C-terminal membrane domain in the stability of yeast Pma1 Hϩ-ATPase and in the delivery of the ATPase to the plasma membrane
Summary
The single most abundant protein in the yeast plasma membrane is a 100-kDa Hϩ-ATPase that accounts for at least 10% of total plasma membrane protein [1]. Studies [11], HA-tagged wild-type ATPase could be detected as early as 15 min after induction of expression, and it was exported efficiently through the secretory pathway to reach the plasma membrane by 60 min post-induction.
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