Description

• Title:
  The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p.
• Creator:
  Flis, Krzysztof;
  Hinzpeter, Alexandre;
  Edelman, Aleksander;
  Kurlandzka, Anna
• Description:
  The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.
• Object availability:
  free access
• Date:
  2005.09.15
• Type:
  Article;
  PeerReviewed
• Language:
  English
• Format:
  application/pdf
• Relation: http://eprints.ibb.waw.pl/395 ;http://www.biochemj.org/bj/390/0655/bj3900655.htm ;doi:10.1042/BJ20050480
• Subject:
  Q Science (General);
  QH Natural history;
  QH426 Genetics;
  QP Physiology
• Identifier: http://eprints.ibb.waw.pl/395/1/KFLIS2005.pdf ;http://eprints.ibb.waw.pl/395/7/KFLIS2005_supplemental_data.pdf ;
  Flis, Krzysztof and Hinzpeter, Alexandre and Edelman, Aleksander and Kurlandzka, Anna (2005) The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p. The Biochemical journal, 390 (Pt 3). pp. 655-64. ISSN 1470-8728
• Data provider:
  Repozytorium Instytutu Biochemii i Biofizyki Polskiej Akademii Nauk
• Can I use it?:
  seek permission
• Type:
  other