Probing the membrane topology of Candida tropicalis cytochrome P450

Détails

ID Serval
serval:BIB_F95EA6FEFA50
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Probing the membrane topology of Candida tropicalis cytochrome P450
Périodique
European Journal of Biochemistry
Auteur(s)
Sanglard  D., Sengstag  C., Seghezzi  W.
ISSN
0014-2956 (Print)
Statut éditorial
Publié
Date de publication
09/1993
Volume
216
Numéro
2
Pages
477-85
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Sep 1
Résumé
The membrane topology of two alkane-inducible cytochromes P450 from the yeast Candida tropicalis, alk1 and alk2, was tested by construction of fusion proteins with part of invertase and histidinol dehydrogenase (invHIS4C) and expression in a Saccharomyces cerevisiae his4 mutant. Depending on the localization of invHIS4C on the endoplasmic reticulum (ER) cytoplasmic or luminal side, the enzyme converts histidinol to histidine and allows the his4 yeast strain to grow on histidinol-supplemented medium. The N-terminal segments of alk1 and alk2 were fused to invHIS4C at three different locations that follow the first alk1 and alk2 transmembrane domains or a second putative transmembrane domain of alk1. The combination of this in vivo assay with subcellular immunoprecipitations of the expressed fusion proteins allowed us to establish that both P450s contain only one transmembrane domain with their N-terminus located in the ER lumen. Deletions performed in these fusion proteins removing the first transmembrane domain of alk1 (delta TM) resulted in a less efficient targeting to the ER membrane but did not prevent their insertion in these membranes. Furthermore deletion of a negatively charged peptide preceding the first alk1 transmembrane domain (delta L) in an invHIS4C protein fused after this domain caused the N-terminal to have a positive net charge and to be oriented in the cytoplasm thus translocating the remaining protein into the ER lumen. The presence of the second hydrophobic segment, however, prevented the complete translocation of this fusion protein into the ER lumen. This study describes the first assessment of P450 membrane topology using an in vivo technique.
Mots-clé
Amino Acid Sequence Base Sequence Candida/*enzymology/genetics Cytochrome P-450 Enzyme System/*chemistry/metabolism DNA, Fungal Fungal Proteins Histidine/biosynthesis Histidinol/metabolism Membrane Proteins/*chemistry/metabolism Molecular Sequence Data Protein Conformation Recombinant Fusion Proteins/chemistry/metabolism Saccharomyces cerevisiae Sequence Homology, Amino Acid Transformation, Genetic
Pubmed
Web of science
Création de la notice
25/01/2008 15:40
Dernière modification de la notice
03/03/2018 22:53
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