Weinberger Adina, Kamena Faustin, Kama Rachel, Spang Anne, Gerst Jeffrey E
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Mol Biol Cell. 2005 Oct;16(10):4918-30. doi: 10.1091/mbc.e05-02-0101. Epub 2005 Aug 10.
Previously, we demonstrated that the phosphorylation of t-SNAREs by protein kinase A (PKA) affects their ability to participate in SNARE complexes and to confer endocytosis and exocytosis in yeast. Here, we show that the presumed phosphorylation of a conserved membrane-proximal PKA consensus site (serine-317) in the Sed5 t-SNARE regulates endoplasmic reticulum (ER)-Golgi transport, as well as Golgi morphology. Sed5 is a phosphoprotein, and both alanine and aspartate substitutions in serine-317 directly affect intracellular protein trafficking. The aspartate substitution results in elaboration of the ER, defects in Golgi-ER retrograde transport, an accumulation of small transport vesicles, and the inhibition of growth of most cell types. In contrast, the alanine substitution has no deleterious effects upon transport and growth, but results in ordering of the Golgi into a structure reminiscent of mammalian apparatus. This structure seems to require the recycling of Sed5, because it was found not to occur in sec21-2 cells that are defective in retrograde transport. Thus, a cycle of Sed5 phosphorylation and dephosphorylation is required for normal t-SNARE function and may choreograph Golgi ordering and dispersal.
此前，我们证明蛋白激酶A（PKA）对t-SNAREs的磷酸化作用会影响其参与SNARE复合体以及在酵母中进行内吞作用和外排作用的能力。在此，我们表明Sed5 t-SNARE中一个保守的膜近端PKA共有位点（丝氨酸-317）的假定磷酸化作用调节内质网（ER）-高尔基体转运以及高尔基体形态。Sed5是一种磷蛋白，丝氨酸-317处的丙氨酸和天冬氨酸替代都会直接影响细胞内蛋白质运输。天冬氨酸替代导致内质网扩张、高尔基体-内质网逆行转运缺陷、小运输囊泡积累以及大多数细胞类型生长受到抑制。相比之下，丙氨酸替代对运输和生长没有有害影响，但会使高尔基体排列成一种类似于哺乳动物细胞器的结构。这种结构似乎需要Sed5的循环利用，因为在逆行转运有缺陷的sec21-2细胞中未发现这种结构。因此，Sed5的磷酸化和去磷酸化循环对于正常的t-SNARE功能是必需的，并且可能编排高尔基体的排列和分散。
