Gaynor E C, Emr S D
Department of Biology, University of California, San Diego, La Jolla 92093-0668, USA.
J Cell Biol. 1997 Feb 24;136(4):789-802. doi: 10.1083/jcb.136.4.789.
The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (gamma-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and alpha-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY-invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi-ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.
外套膜蛋白（COPI）复合体介导含有双赖氨酸回收基序的膜蛋白从高尔基体到内质网的循环利用。然而，COPI最初被表征为一种顺向作用的包被复合体。为了全面研究COPI在ER/高尔基体运输以及分泌途径中的直接和主要作用，我们利用基于PCR的诱变技术在酿酒酵母中产生了一个COPI基因SEC21（γ-COP）的新的温度条件突变等位基因。出乎意料的是，所有新的sec21温度敏感型突变体都表现出显著的、货物选择性的从内质网到高尔基体的运输缺陷。在这些突变体中，几种蛋白质（即羧肽酶Y和α-因子）在非允许温度下完全被困在内质网中；然而，这些以及其他COPI突变体中的其他蛋白质（即蔗糖酶和热休克蛋白150）却能正常分泌。布雷菲德菌素A也诱导出了几乎相同的货物特异性内质网到高尔基体的运输缺陷。相比之下，所有测试的蛋白质从内质网到高尔基体的运输都需要COPII（内质网到高尔基体的包被复合体）、Sec18p（N-乙基马来酰亚胺敏感因子）和Sec22p（囊泡-可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）。这些数据共同表明，COPI在顺向运输中起着关键但间接的作用，可能是通过指导将某些货物包装到内质网到高尔基体的COPII囊泡中所需的运输因子的回收。有趣的是，羧肽酶Y-蔗糖酶杂合蛋白，像蔗糖酶而不像羧肽酶Y，逃脱了sec21温度敏感型突变体的内质网阻滞，这表明包装到COPII囊泡中可能由货物蛋白自身的顺式作用分选决定簇介导。这些杂合蛋白被有效地靶向到液泡，表明COPI对于从高尔基体到液泡的调节运输也不是直接必需的。此外，sec21突变体在非允许温度下在早期高尔基体区室表现出早期高尔基体特异性糖基化缺陷和结构畸变，但晚期高尔基体区室没有。这些研究共同表明，尽管COPI在高尔基体-内质网回收以及顺式高尔基体的维持/功能中起着重要且很可能是直接的作用，但COPI似乎不是通过分泌途径进行顺向运输所直接必需的。
