Institute of Biology, Department of Plant Physiology, Humboldt University, 10115 Berlin, Germany.
Mol Plant. 2012 Jan;5(1):43-62. doi: 10.1093/mp/ssr048. Epub 2011 Jul 10.
Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUT1-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane (DRM) fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiquitin system (SUS) to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane-associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUT1-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was observed between the StSUT1-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUT1-interacting proteins, a protein disulfide isomerase (PDI), interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.
蛋白质复合物的组织对于许多细胞功能至关重要。最近，SUT1 蛋白被证明形成同源二聚体复合物，与酵母、植物中的脂筏样微区相关，并在受到布雷菲德菌素 A 作用时发生内吞作用。因此，我们旨在鉴定可能参与 SUT1 二聚化、内吞作用或靶向筏样微区的 SUT1 相互作用蛋白。因此，我们鉴定了与去污剂抗性膜 (DRM) 部分相关的马铃薯膜蛋白。在所鉴定的蛋白中，我们明确证实了 StSUT1 是马铃薯源叶 DRM 的一部分。我们使用酵母双杂交分裂泛素系统 (SUS) 系统地筛选蔗糖转运蛋白 StSUT1 与体内其他膜相关或可溶性蛋白之间的相互作用。SUS 筛选后，使用亲和纯化的 StSUT1 特异性肽抗体进行免疫沉淀，并对共沉淀蛋白进行质谱分析。在共免疫沉淀和去污剂抗性膜部分中鉴定的 StSUT1 相互作用蛋白之间观察到很大的重叠。SUT1 相互作用蛋白之一，即蛋白质二硫键异构酶 (PDI)，也与其他蔗糖转运蛋白相互作用。讨论了 PDI 作为护送蛋白的潜在作用。
