Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Mol Biol Cell. 2011 Jun 1;22(11):1919-29. doi: 10.1091/mbc.E10-10-0800. Epub 2011 Apr 6.
Intracellular trafficking of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by amino acid abundance. When amino acids are scarce Gap1p is sorted to the plasma membrane, whereas when amino acids are abundant Gap1p is sorted from the trans-Golgi through the multivesicular endosome (MVE) and to the vacuole. Here we test the hypothesis that Gap1p itself is the sensor of amino acid abundance by examining the trafficking of Gap1p mutants with altered substrate specificity and transport activity. We show that trafficking of mutant Gap1p(A297V), which does not transport basic amino acids, is also not regulated by these amino acids. Furthermore, we have identified a catalytically inactive mutant that does not respond to complex amino acid mixtures and constitutively sorts Gap1p to the plasma membrane. Previously we showed that amino acids govern the propensity of Gap1p to recycle from the MVE to the plasma membrane. Here we propose that in the presence of substrate the steady-state conformation of Gap1p shifts to a state that is unable to be recycled from the MVE. These results indicate a parsimonious regulatory mechanism by which Gap1p senses its transport substrates to set an appropriate level of transporter activity at the cell surface.
酵母的一般氨基酸通透酶 Gap1p 的细胞内运输受氨基酸丰度的调节。当氨基酸匮乏时，Gap1p 被分拣到质膜，而当氨基酸丰富时，Gap1p 通过多泡体（MVE）从高尔基体分拣到液泡。在这里，我们通过检查改变底物特异性和运输活性的 Gap1p 突变体的运输情况，来检验 Gap1p 本身是氨基酸丰度的传感器这一假说。我们表明，不运输碱性氨基酸的突变体 Gap1p(A297V)的运输也不受这些氨基酸的调节。此外，我们已经鉴定出一种无催化活性的突变体，它对复杂的氨基酸混合物没有反应，并且使 Gap1p 持续分拣到质膜。之前我们表明，氨基酸控制 Gap1p 从 MVE 再循环到质膜的倾向。在这里，我们提出，在存在底物的情况下，Gap1p 的稳态构象向一种无法从 MVE 再循环的状态转变。这些结果表明了一种简单的调节机制，通过该机制，Gap1p 感知其转运底物，在细胞表面设定适当的转运体活性水平。
