Functional analysis of putative phosphoenolpyruvate transporters localized to the Golgi apparatus in Schizosaccharomyces pombe.
Functional analysis of putative phosphoenolpyruvate transporters localized to the Golgi apparatus in Schizosaccharomyces pombe.
复制标题
粟酒裂殖酵母高尔基体中假定的磷酸烯醇丙酮酸转运蛋白的功能分析。
DOI：
10.1111/1567-1364.12207
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发表时间：
2014
期刊：
影响因子：
3.2
通讯作者：
Takegawa K
中科院分区：
文献类型：
--
作者：
Yoritsune KI;Higuchi Y;Matsuzawa T;Takegawa K
裂殖酵母细胞表面带负电荷是由于存在丙酮化低聚糖，这对细胞-细胞识别是重要的。然而，丙酮酸向低聚糖供应的机制尚不清楚。在这里，我们分析了三个假定的磷酸烯醇式丙酮酸(PEP)转运蛋白基因(pet1+，pet2+和pet3+)INS。Pombe，通过与拟南芥PEP转运蛋白AtPPT1的序列同源性搜索而鉴定。裂殖酵母Pombe菌株携带中断的pEt1+(Pet1Δ)或inpet2+(Pet2Δ)，而不携带中断的pEt3+(Pet3Δ)的菌株，显示细胞表面丙酮酸水平降低。通过在相应的干扰物中表达标记了绿色荧光蛋白(GFP)的Pet1p和Pet2p，丙酮酸水平的下降恢复到了对照水平。荧光显微镜研究表明，GFP标记的Pet1p和Pet2p定位于高尔基体。虽然AtPPT1和AtPPT2的表达都没有抑制Pet1的Δ表型，但嵌合结构的表达，其中AtPPT1和AtPPT2的N端区域被Pet1p的N端区域取代，部分抑制了Pet1的Δ表型。此外，用重组pvg1p和PEP孵育PET1Δ细胞后，细胞表面负电荷的减少得以恢复。因此，Pet1p和Pet2p可能参与了PEP从细胞质运输到高尔基体的过程。
The cell surface ofSchizosaccharomyces pombeis negatively charged due to the presence of pyruvylated oligosaccharides, which is important for cell–cell recognition. However, the mechanism of pyruvate supply to oligosaccharides is not clearly understood. Here, we analyzed three putative phosphoenolpyruvate (PEP) transporter genes (pet1+,pet2+, andpet3+) inS. pombe, identified by sequence homology search against theArabidopsis thalianaPEP transporter AtPPT1.Schizosaccharomyces pombestrain carrying a disruption inpet1+(pet1Δ) or inpet2+(pet2Δ), but not the strain carrying a disruption inpet3+(pet3Δ), showed reduced pyruvate level on the cell surface. This reduction in pyruvate level was restored to the control level by expressing green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants. Fluorescence microscope studies revealed that GFP-tagged Pet1p and Pet2p were localized to the Golgi apparatus. Although expression of neither AtPPT1 nor AtPPT2 suppressed thepet1Δ phenotype, that of chimeric constructs, where the N-terminal regions of AtPPT1 and AtPPT2 were replaced by the N-terminal region of Pet1p, partially suppressed thepet1Δ phenotype. Furthermore, the reduction in cell surface negative charge inpet1Δ cells was restored by incubating these cells with recombinant Pvg1p and PEP. Thus, Pet1p and Pet2p are likely involved in transporting PEP from the cytoplasm into the Golgi.