Instituto de Biología Funcional y Genómica and Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, Salamanca, Spain.
Mol Microbiol. 2013 Oct;90(2):252-66. doi: 10.1111/mmi.12360. Epub 2013 Aug 26.
Chs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP-1-dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N-terminal cytosolic region. However, the truncated (Δ126)Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI- and Rer1-dependent manner. A subset of the oligomerization-deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N-terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly.
Chs3 是酿酒酵母几丁质合成酶 III 的催化亚基，是一种复杂的多跨膜蛋白，其质膜表达受到严格控制：从内质网输出需要与 Chs7 相互作用；从高尔基体的输出依赖于外切体复合物，而精确的芽颈定位依赖于内吞作用。此外，Chs3 能够以 AP-1 依赖性的方式从内体有效地回收至 TGN。在这里，我们发现 Chs3 的输出需要货物受体 Erv14 的参与，这一步骤不依赖于 Chs7。Chs3 在 ER 中通过其 N 端胞质区域寡聚化。然而，截短的（Δ126）Chs3 仍能被 Erv14 输出，但以 COPI 和 Rer1 依赖性的方式从高尔基体返回 ER。一部分寡聚化缺陷的 Chs3 蛋白逃避了高尔基体的质量控制并到达质膜，在那里它们具有酶活性但内吞作用较差。这导致 CSIII 水平升高，但 Calcofluor White 抗性降低，这可以解释为 Calcofluor White 插入新生几丁质纤维之间的减少。我们的数据表明，Chs3 通过其 N 端的寡聚化对于正确的蛋白质运输和几丁质合成是必需的，因此在细胞内受到监测。
