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Title

Functional role of highly conserved residues of the N-terminal tail and first transmembrane segment of a P4-ATPase

AuthorsPerandrés-López, R.; Sánchez-Cañete, M.P.; Gamarro, F.; Castanys, S.
Issue Date2018
PublisherPortland Press
CitationBiochemical Journal 475: 887- 899 (2018)
AbstractThe P4 family of P-type ATPases (P4-ATPases) plays an important role in maintaining phospholipid asymmetry in eukaryotic cell membranes. Leishmania miltefosine transporter (LMT) is a plasma membrane (PM) P4-ATPase that catalyses translocation into the parasite of the leishmanicidal drug miltefosine as well as phosphatidylcholine and phosphatidylethanolamine analogues. In the present study, we analysed the role, in LMT, of a series of highly conserved amino acids previously undescribed in the N-terminal region of P4-ATPases. Seven residues were identified and, according to an LMT structural model, five were located in the cytosolic N-terminal tail (Asn58, Ile60, Lys64, Tyr65 and Phe70) and the other two (Pro72 and Phe79) in the first transmembrane segment (TM1). Alanine-scanning mutagenesis analysis showed that N58A, Y65A and F79A mutations caused a considerable reduction in the LMT translocase activity. These mutations did not affect protein expression levels. We generated additional mutations in these three residues to assess the influence of the conservation degree on LMT translocase activity. Some of these mutations reduced expression levels without affecting the interaction between LMT and its CDC50 subunit, LRos3. Conserved and non-conserved mutations in the invariant residue Asn58 drastically reduced the translocase activity. Consequently, Asn58 may be necessary to achieve optimal catalytic LMT activity as previously described for the potentially equivalent Asn39 of the sarco/endoplasmic reticulum Ca-ATPase isoform 1a (SERCA1a). Additionally, conservation of a hydrophobic residue at position 79 is crucial for LMT stability.
Publisher version (URL)http://dx.doi.org/10.1042/BCJ20170749
URIhttp://hdl.handle.net/10261/212230
Identifiersdoi: 10.1042/BCJ20170749
e-issn: 1470-8728
issn: 0264-6021
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