Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase
Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase
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DOI：
10.1074/jbc.m601818200
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发表时间：
2006-08-18
影响因子：
4.8
通讯作者：
Slayman, Carolyn W.
中科院分区：
文献类型：
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作者：
Mason, A. Brett;Allen, Kenneth E.;Slayman, Carolyn W.
在P型阳离子转运ATP酶的大家族中，成员的C-末端跨膜螺旋的数目不同，从Cu 2 +-ATP酶中的2个到H+-、Na+、K+-、Mg 2 +-和Ca 2 +-ATP酶中的6个。在这项研究中，酵母Pma 1 H+-ATP酶已作为一个模型，以检查的C-末端膜结构域在ATP酶的稳定性和靶向质膜的作用。从主要胞质环的中间到延伸的胞质尾的中间构建连续截短，一次一个地添加回C末端跨膜螺旋。当所得到的构建体在酵母中瞬时表达时，半衰期从Pma 1 Delta 452中的70分钟稳定增加到Pma 1 Delta 901中的348分钟，但即使是最长的构建体也比野生型ATP酶（t（1/2）= 11 h）稳定性差得多。共聚焦免疫荧光显微镜显示，12个结构中的11个在内质网中被捕获并在蛋白酶体中降解。唯一一个逃脱ER的截短型ATP酶Pma 1 Delta 901缓慢地行进到质膜，在那里它水解ATP并支持生长。然而，有限的胰蛋白酶溶解显示Pma 1 Delta 901错误折叠，导致过早递送到空泡中降解。作为模型底物，这一系列的截短肯定了整个C-末端结构域对酵母H+-ATP酶生物合成的重要性，并定义了羧基尾中20个氨基酸的序列元件，这对ER逃逸和运输到质膜至关重要。
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 Delta 452 to 348 min in Pma1 Delta 901, 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 Delta 901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 Delta 901 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.