MATE1 has an external COOH terminus, consistent with a 13-helix topology

Xiaohong Zhang, Stephen H. Wright

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

The mammalian members of the Multidrug And Toxin Extruder family, i.e., MATE1 and MATE2-K, are suspected of mediating the luminal step in renal secretion of organic cations. The 1,000+ prokaryotic/fungal/plant MATE family members are predicted to have 12 transmembrane helices (TMHs), whereas MATE1/2-K appear to have an additional (13th) COOH-terminal helix. Here, we determined whether rabbit MATE1 has an external COOH terminus, consistent with the presence of 13 TMHs. A V5 epitope tag at the COOH terminus of MATE1 was freely accessible to external V5 antibody, whereas tags at the NH2 terminus, or at sites of truncation within the long cytoplasmic loop between predicted TMHs 12 and 13, were only accessible to the V5 antibody following permeabilization of the membrane. The truncated mutants that lacked TMH13 still retained transport activity, indicating that the terminal helix was not necessary for transport function. Cells that expressed a mutant lacking only TMH13 displayed similar Kt and Jmax values to those of the full-length protein, although when normalized to protein expressed at the plasma membrane, the transport rate of the mutant was <10% that of full-length MATE1. An effectively cysteine-less MATE1 mutant (Δ13Cys) was functional and refractory to reaction with the impermeant marker of accessible cysteine residues, maleimide-PEO2-biotin. Δ13Cys mutants with an added cysteine residue at the truncation sites within the terminal cytoplasmic loop reacted with maleimide biotin only after permeabilization of the membrane, whereas a mutant with a cysteine residue at the COOH terminus was freely accessible to maleimide biotin. These data are consistent with a mammalian MATE topology that includes 13 TMHs and indicate that the terminal TMH, although not necessary for transport function, may influence the turnover characteristics of the transporter.

Original languageEnglish (US)
Pages (from-to)F263-F271
JournalAmerican Journal of Physiology - Renal Physiology
Volume297
Issue number2
DOIs
StatePublished - Aug 2009

Keywords

  • Kidney
  • Luminal
  • Organic cation
  • Proximal tubule
  • Transport

ASJC Scopus subject areas

  • Physiology
  • Urology

Fingerprint Dive into the research topics of 'MATE1 has an external COOH terminus, consistent with a 13-helix topology'. Together they form a unique fingerprint.

Cite this