P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.
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Research Article|
May 27 2016
Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase
Sara R. Costa;
Sara R. Costa
*Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark
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Magdalena Marek;
Magdalena Marek
1
*Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark
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Kristian B. Axelsen;
Kristian B. Axelsen
*Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark
†SIB Swiss Institute of Bioinformatics, CMU, CH-1211, Geneva, Switzerland
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Lisa Theorin;
Lisa Theorin
*Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark
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Thomas G. Pomorski;
Thomas G. Pomorski
*Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark
‡Faculty of Chemistry and Biochemistry, Department of Molecular Biochemistry, Ruhr University Bochum, Universitätstrasse 150, D-44780 Bochum, Germany
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Rosa L. López-Marqués
Rosa L. López-Marqués
2
*Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark
2To whom correspondence should be addressed (email rlo@plen.ku.dk).
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Publisher: Portland Press Ltd
Received:
March 09 2016
Revision Received:
April 01 2016
Accepted:
April 04 2016
Accepted Manuscript online:
April 05 2016
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society
2016
Biochem J (2016) 473 (11): 1605–1615.
Article history
Received:
March 09 2016
Revision Received:
April 01 2016
Accepted:
April 04 2016
Accepted Manuscript online:
April 05 2016
Citation
Sara R. Costa, Magdalena Marek, Kristian B. Axelsen, Lisa Theorin, Thomas G. Pomorski, Rosa L. López-Marqués; Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase. Biochem J 1 June 2016; 473 (11): 1605–1615. doi: https://doi.org/10.1042/BCJ20160207
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