Hutchins M U, Klionsky D J
Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2001 Jun 8;276(23):20491-8. doi: 10.1074/jbc.M101150200. Epub 2001 Mar 22.
One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. alpha-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.
真核细胞面临的一个挑战是蛋白质在翻译后导入细胞器。当蛋白质组装成大复合物时，这个问题会更加严重。氨肽酶I（API）是酿酒酵母液泡/溶酶体中的一种驻留水解酶。API的前体形式在细胞质中组装成十二聚体，并在导入过程中维持这种寡聚形式。API前体形式的液泡递送需要一种称为细胞质到液泡靶向（Cvt）途径的囊泡机制。Cvt途径的许多成分也用于降解性自噬途径。α-甘露糖苷酶（Ams1）是另一种独立于分泌途径进入液泡的驻留水解酶；然而，其液泡递送机制尚未确定。我们发现，在Cvt和自噬途径有缺陷的突变体中，Ams1的液泡定位被阻断。我们发现Ams1在细胞质中形成寡聚体。在囊泡分解受阻的菌株的液泡下小泡中也检测到Ams1的寡聚形式，这表明它在导入过程中保持其寡聚形式。这些结果确定Ams1是Cvt和自噬途径的第二种生物合成货物蛋白。
