title: The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.
abstract: Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity.
authors.affiliation: University of Duisburg-Essen, Department of Endocrinology and Metabolism and Division of Laboratory Research, Hufelandstraße 55, 45147 Essen, Germany. Electronic address: denise.zwanziger@uk-essen.de.
authors.forename: Denise
authors.initials: D
authors.lastname: Zwanziger
authors.affiliation: University of Duisburg-Essen, Department of Endocrinology and Metabolism and Division of Laboratory Research, Hufelandstraße 55, 45147 Essen, Germany. Electronic address: mathias.schmidt1987@gmx.de.
authors.forename: Mathias
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Charitè-Berlin, Institute of Experimental Pediatric Endocrinology, Augustenburgerplatz 1, 13353 Berlin, Germany. Electronic address: jana.fischer@charite.de.
authors.forename: Jana
authors.initials: J
authors.lastname: Fischer
authors.affiliation: Charitè-Berlin, Institute of Experimental Pediatric Endocrinology, Augustenburgerplatz 1, 13353 Berlin, Germany. Electronic address: gunnar.kleinau@charite.de.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Kleinau
authors.affiliation: Rheinische Friedrich-Wilhelms Universität Bonn, Institut für Biochemie und Molekularbiologie, Nussallee 11, 53115 Bonn, Germany. Electronic address: doreen.braun@uni-bonn.de.
authors.forename: Doreen
authors.initials: D
authors.lastname: Braun
authors.affiliation: Rheinische Friedrich-Wilhelms Universität Bonn, Institut für Biochemie und Molekularbiologie, Nussallee 11, 53115 Bonn, Germany. Electronic address: uschweiz@uni-bonn.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schweizer
authors.affiliation: University of Duisburg-Essen, Department of Endocrinology and Metabolism and Division of Laboratory Research, Hufelandstraße 55, 45147 Essen, Germany. Electronic address: lars.moeller@uni-due.de.
authors.forename: Lars Christian
authors.initials: LC
authors.lastname: Moeller
authors.affiliation: Charitè-Berlin, Institute of Experimental Pediatric Endocrinology, Augustenburgerplatz 1, 13353 Berlin, Germany. Electronic address: heike.biebermann@charite.de.
authors.forename: Heike
authors.initials: H
authors.lastname: Biebermann
authors.affiliation: University of Duisburg-Essen, Department of Endocrinology and Metabolism and Division of Laboratory Research, Hufelandstraße 55, 45147 Essen, Germany. Electronic address: dagmar.fuehrer@uk-essen.de.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Fuehrer
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D004285:Dogs; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D027501:Monocarboxylic Acid Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D027981:Symporters; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D027501:Monocarboxylic Acid Transporters; C090735:SLC16A2 protein, human; D027981:Symporters; D025801:Ubiquitin; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine
keywords: Degradation; MCT8; N-terminus; Oligomerization; Ubiquitination