title: Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase.
abstract: 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, Asn(181) and Asn(231) Whereas mutation of Asn(231) seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn(181) disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys(86) and Cys(107) 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 Cys(158) and Cys(172) 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.
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark.
authors.forename: Sara R
authors.initials: SR
authors.lastname: Costa
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Marek
authors.affiliation: 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.
authors.forename: Kristian B
authors.initials: KB
authors.lastname: Axelsen
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark.
authors.forename: Lisa
authors.initials: L
authors.lastname: Theorin
authors.affiliation: 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.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Pomorski
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark rlo@plen.ku.dk.
authors.forename: Rosa L
authors.initials: RL
authors.lastname: López-Marqués
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains / Q000235:genetics / Q000502:physiology; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D014026:Nicotiana / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D021122:Protein Subunits; D001216:Asparagine; D000251:Adenosine Triphosphatases; D003545:Cysteine
keywords: Arabidopsis thaliana; Cdc50 ectodomain; N-glycosylation; disulfide bond; flippase