Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
J Biol Chem. 2012 May 18;287(21):17801-17811. doi: 10.1074/jbc.M112.345058. Epub 2012 Mar 26.
In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.
在异源和内源性表达系统中，我们研究了内质网蛋白 44（ERp44）及其复合伴侣内质网氧化酶 1-α（Ero1-Lα）在调节血清素转运体（SERT）二硫键形成机制中的作用，SERT 是一种寡聚糖蛋白。ERp44 是一种内质网腔伴侣蛋白，有利于二硫键连接的寡聚蛋白的成熟。ERp44 通过与 Ero1-Lα 结合，在蛋白质从内质网释放中发挥关键作用。硫氧还蛋白样结构域的突变会阻碍 ERp44C29S 与 SERT 的结合，SERT 上的第二外环有三个半胱氨酸残基（Cys-200、Cys-209 和 Cys-109）。我们通过 shRNA 敲低 ERp44 和 Ero1-Lα 进一步探讨了蛋白质伴侣的作用。这些努力导致 SERT 向质膜的定位增加，但 5-羟色胺（5-HT）摄取率降低，表明 ERp44 保留机制在 SERT 蛋白的正确成熟中的重要性。这些数据得到了从 ERp44 shRNA 细胞上的 SERT 用 N-生物素氨基乙基甲硫磺酸酯（MTSEA-biotin）标记获得的数据的强烈支持。MTSEA-biotin 仅与质膜外相的游离半胱氨酸残基相互作用。有趣的是，似乎 SERT 的 Cys-200 和 Cys-209 在 ERp44 沉默的细胞中可被 MTSEA-biotin 标记。然而，在对照细胞中，这些 Cys 残基被占据，并用 MTSEA-biotin 标记的量较少。此外，与野生型相比，ERp44 优先与 SERT 突变体（C200S、C209S 和 C109A）结合。这些与伴侣蛋白的相互作用可能反映了 Cys-200 和 Cys-209 SERT 突变体不能形成二硫键和自身缔合的能力，这一点可以通过免疫沉淀测定得到证明。基于这些综合发现，我们假设 ERp44 与 Ero1-Lα 一起在 SERT 的二硫键形成中发挥重要作用，这可能是 SERT 分子形成寡聚形式的组装的前提步骤。
