Shaw Jonathan D, Hama Hiroko, Sohrabi Farrokh, DeWald Daryll B, Wendland Beverly
Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD, 21218, USA.
Traffic. 2003 Jul;4(7):479-90. doi: 10.1034/j.1600-0854.2003.t01-1-00106.x.
The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P2], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P2 production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P2 is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.
内吞途径将货物从质膜运输到早期内体，在早期内体中，某些货物被分选到晚期内体/多囊泡体。运往溶酶体的生物合成货物也通过多囊泡体进行运输。一旦被运送到多囊泡体，运往溶酶体内部的货物会被选择性地分选到芽生到多囊泡体腔中的小泡中。当多囊泡体与溶酶体的限制膜融合时，这些小泡会释放到溶酶体腔中。酵母蛋白Fab1催化磷脂酰肌醇（3,5）二磷酸[PtdIns(3,5)P2]的产生，它对于多囊泡体中生物合成货物的正确分选是必需的。利用内吞筛选，我们分离出了FAB1的一个新等位基因，该等位基因在脂质激酶结构域中含有一个点突变。对这个等位基因的表征揭示了PtdIns(3,5)P2产生减少、液泡形态改变以及生物合成蛋白分选缺陷。我们还发现质膜蛋白Ste3的内吞在内化步骤的下游部分受阻，并且在fab1突变体中，染料FM4-64向液泡的运输延迟。此外，在fab1突变体中，Ste3不能有效地分选到多囊泡体小泡中，而是定位于液泡的限制膜。这些数据表明PtdIns(3,5)P2对于通过晚期内体/多囊泡体进行内吞货物的正确运输和分选是必需的。
