Universität zu Köln

Tschirka, Julia, Bach, Markus, Kisis, Ilmars, Lemmen, Julia, Gnoth, Mark Jean and Gruendemann, Dirk (2020). Transporter tandems: precise tools for normalizing active transporter in the plasma membrane. Biochem. J., 477 (21). S. 4191 - 4207. LONDON: PORTLAND PRESS LTD. ISSN 1470-8728

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Abstract

The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC-MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher ( f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification - as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Tschirka, Julia UNSPECIFIED UNSPECIFIED UNSPECIFIED Bach, Markus UNSPECIFIED UNSPECIFIED UNSPECIFIED Kisis, Ilmars UNSPECIFIED UNSPECIFIED UNSPECIFIED Lemmen, Julia UNSPECIFIED UNSPECIFIED UNSPECIFIED Gnoth, Mark Jean UNSPECIFIED UNSPECIFIED UNSPECIFIED Gruendemann, Dirk UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-313538
DOI:	10.1042/BCJ20200666
Journal or Publication Title:	Biochem. J.
Volume:	477
Number:	21
Page Range:	S. 4191 - 4207
Date:	2020
Publisher:	PORTLAND PRESS LTD
Place of Publication:	LONDON
ISSN:	1470-8728
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language EXTRANEURONAL MONOAMINE TRANSPORTER; GLUT1 GLUCOSE-TRANSPORTER; MASS-SPECTROMETRY; DRUG TRANSPORTERS; GLYCEROL-3-PHOSPHATE TRANSPORTER; UDP-GLUCURONOSYLTRANSFERASES; PROTEOMIC ANALYSIS; ESCHERICHIA-COLI; QUANTIFICATION; EXPRESSION Multiple languages Biochemistry & Molecular Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/31353