Chen Linyi, Davis Nicholas G
Department of Physiology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA.
Traffic. 2002 Feb;3(2):110-23. doi: 10.1034/j.1600-0854.2002.030204.x.
The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway links to vacuolar degradation of the receptor, while a ligand-dependent uptake pathway links primarily to recycling and thus, receptor reutilization. Ste3p ubiquitination triggers its uptake into the constitutive pathway. The present work considers the role of the receptor ubiquitination associated with the Ste3p ligand-dependent endocytosis mechanism. The doa4delta mutation which reduces the cellular availability of ubiquitin blocks the Ste3p constitutive uptake. Uptake into the Ste3p ligand-dependent recycling pathway, however, continues unimpaired. The ubiquitin independence of Ste3p ligand-dependent uptake was further indicated by analysis of receptor mutants having Lys-to-Arg substitutions at all possible ubiquitin acceptor sites. Again, the ligand-induced internalization was unimpaired. Furthermore, no discernible effect was seen on either recycling or on the slow PEP4-dependent turnover of the receptor (for receptor internalized via the ligand-dependent mechanism, trafficking to the vacuole/lysosome is the minor, alternate fate to recycling). However, one striking effect of the Lys-to-Arg mutations was noted. Following a prolonged exposure of the cells to the a-factor ligand, rather than being delivered to the vacuolar lumen, the Lys-to-Arg receptor was found to localize instead to the limiting membrane of the vacuole. Thus, while receptor ubiquitination clearly is not required for either the a-factor-dependent uptake into recycling pathway or for the recycling itself, it does affect the routing of receptor to the vacuole, likely by affecting the routing through the late endosomal, multivesicular body: ubiquitinated receptor may be selected into the internal, lumenal vesicles, while unmodified receptor may be left to reside at the limiting external membrane.
酵母a因子受体（Ste3p）存在两种机制不同的内吞模式：一种是组成型、不依赖配体的途径，与受体的液泡降解相关；另一种是依赖配体的摄取途径，主要与再循环相关，从而实现受体的再利用。Ste3p泛素化触发其进入组成型途径。本研究探讨了与Ste3p依赖配体内吞机制相关的受体泛素化的作用。降低细胞内泛素可用性的doa4delta突变会阻断Ste3p的组成型摄取。然而，进入Ste3p依赖配体再循环途径的摄取不受影响。通过分析在所有可能的泛素接受位点具有赖氨酸到精氨酸替代的受体突变体，进一步表明了Ste3p依赖配体摄取不依赖泛素。同样，配体诱导的内化不受影响。此外，对再循环或受体缓慢的依赖PEP4的周转均未观察到明显影响（对于通过依赖配体机制内化的受体，转运到液泡/溶酶体是再循环的次要替代命运）。然而，注意到赖氨酸到精氨酸突变有一个显著影响。在细胞长时间暴露于a因子配体后，发现赖氨酸到精氨酸受体不是被递送到液泡腔，而是定位在液泡的限制膜上。因此，虽然受体泛素化显然对于依赖a因子摄取进入再循环途径或再循环本身不是必需的，但它确实影响受体向液泡的转运，可能是通过影响通过晚期内体、多囊泡体的转运：泛素化的受体可能被选入内部的腔内小泡，而未修饰的受体可能留在限制的外膜上。
