Nicolson T, Conradt B, Wickner W
Molecular Biology Institute and Department of Biological Chemistry, University of Los Angeles, California 90024, USA.
J Bacteriol. 1996 Jul;178(14):4047-51. doi: 10.1128/jb.178.14.4047-4051.1996.
Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle, vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of a soluble inhibitory factor. When mixed with wild-type cytosol, vac5-1 cytosol inhibits the activity of wild-type cytosol. vac5-1 cytosol treated with trypsin or papain is still able to inhibit the activity of Aid-type cytosol. Partial fractionation of vac5-1 cytosol reveals that the protein traction (G25 void volume) can promote fusion if wild-type small molecules are included in the fusion reaction. In contrast, the vac5-l small-molecule fraction retains the full ability to inhibit fusion. Thus, the vac5-1 allele of PHO80 induces the synthesis of a small molecule that is an inhibitor of vacuole inheritance.
液泡向酵母芽中投射出囊泡流和膜性小管，它们在芽中融合，形成子细胞的细胞器。vac5 - 1编码截短形式的Pho80细胞周期蛋白，它会抑制正常的液泡遗传。一种测量液泡融合的体外遗传分析方法可作为该过程几个步骤的模型。我们发现，在体外分析中，从vac5 - 1突变体中分离出的胞质溶胶无法促进野生型液泡的融合。如果野生型液泡与vac5 - 1胞质溶胶和ATP预孵育，它们会以时间和温度依赖的方式不可逆地失活，这表明存在一种可溶性抑制因子。当与野生型胞质溶胶混合时，vac5 - 1胞质溶胶会抑制野生型胞质溶胶的活性。用胰蛋白酶或木瓜蛋白酶处理过的vac5 - 1胞质溶胶仍然能够抑制野生型胞质溶胶的活性。对vac5 - 1胞质溶胶进行部分分级分离发现，如果在融合反应中加入野生型小分子，蛋白质部分（G25空体积）可以促进融合。相反，vac5 - 1小分子部分则完全保留抑制融合的能力。因此，PHO80的vac5 - 1等位基因诱导合成了一种小分子，它是液泡遗传的抑制剂。
