title: The role of ERp44 in maturation of serotonin transporter protein.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Samuel
authors.initials: S
authors.lastname: Freyaldenhoven
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Yicong
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Arif M
authors.initials: AM
authors.lastname: Kocabas
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Enrit
authors.initials: E
authors.lastname: Ziu
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Serra
authors.initials: S
authors.lastname: Ucer
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Raman
authors.initials: R
authors.lastname: Ramanagoudr-Bhojappa
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
authors.forename: Grover P
authors.initials: GP
authors.lastname: Miller
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205. Electronic address: kilicfusun@uams.edu.
authors.forename: Fusun
authors.initials: F
authors.lastname: Kilic
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D005260:Female; D020868:Gene Silencing; D006801:Humans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010088:Oxidoreductases; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; C451899:ERP44 protein, human; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C405327:ERO1A protein, human; D010088:Oxidoreductases