The Role of ERp44 in Maturation of Serotonin Transporter Protein

• Published in: The Journal of biological chemistry Vol. 287; no. 21; pp. 17801 - 17811
• Main Authors: Freyaldenhoven, Samuel, Li, Yicong, Kocabas, Arif M., Ziu, Enrit, Ucer, Serra, Ramanagoudr-Bhojappa, Raman, Miller, Grover P., Kilic, Fusun
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.05.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Cell Line; Cell Membrane - genetics; Cell Membrane - metabolism; Disulfide; Disulfides - metabolism; Endoplasmic Reticulum (ER); Female; Gene Silencing; Humans; Membrane Glycoproteins; Membrane Proteins; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Cell Biology; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Mutation; Oxidoreductases; Post-translational Modification; Protein Multimerization - physiology; Protein Structure, Tertiary; Serotonin Plasma Membrane Transport Proteins - genetics; Serotonin Plasma Membrane Transport Proteins - metabolism; Signal Transduction; Membrane Proteins; Post-translational Modification; Molecular Cell Biology; Disulfide; Endoplasmic Reticulum (ER)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.345058

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. ERp44 favors maturation of disulfide-linked oligomeric proteins. ERp44 bound to SERT but preferentially to Cys mutants; in ERp44-silenced cells, 5-HT uptake was down-regulated; MTSEA-biotin labeled SERT with a higher affinity, indicating more free Cys on SERT in silenced cells. A disulfide link between Cys-200 and Cys-209 is a prerequisite for SERT oligomerization. This is the first study showing the involvement of ERp44 in maturation of SERT.