Luo W j, Chang A
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Mol Biol Cell. 2000 Feb;11(2):579-92. doi: 10.1091/mbc.11.2.579.
The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37 degrees C. Several vps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, and vps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36 cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 and vps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (and vps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface in vps1 cells. We hypothesize that in vps8 and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.
由PMA1编码的质膜ATP酶通过分泌途径被转运至细胞表面。此前，我们鉴定了一个温度敏感型pma1突变体，在该突变体中，新合成的Pma1-7不会被转运至质膜，而是在37℃时错误定位于液泡。几个在液泡蛋白分选方面存在缺陷的vps突变体，通过允许突变型Pma1再次转运至质膜，抑制了靶向缺陷型pma1。在本研究中，我们通过监测Pma1-7在vps36、vps1和vps8突变体中的转运情况，分析了内体系统中的运输过程。诱导表达后，突变型Pma1在vps36细胞的前液泡区室中积累。追踪后，一部分新合成的Pma1-7被转运至质膜。在vps1和vps8细胞中，新合成的突变型Pma1在到达细胞表面之前出现在小的点状结构中。然而，生物合成膜运输在vps8和vps1中似乎遵循不同的途径：液泡蛋白分选受体Vps10p在vps8中稳定，但在vps1中不稳定。此外，通过大量膜标记物FM 4-64的内吞作用发现，vps8（和vps36）存在内吞运输至液泡的缺陷，而vps1则没有。此外，在vps8细胞中，交配受体Ste3从细胞表面的下调存在缺陷，这与受体从内体区室持续循环至质膜一致。这些数据支持了一个模型，即突变型Pma1在vps1细胞中从高尔基体转向表面。我们推测，与vps1相反，在vps8和vps36中，突变型Pma1通过内体中间体转运至表面，这意味着存在一条从内体到表面的运输途径。
