{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T05:12:59Z","timestamp":1791349979411,"version":"4.3.1"},"reference-count":42,"publisher":"Portland Press Ltd.","issue":"11","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2016,6,1]]},"abstract":"<jats:p>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 \u03b2-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 \u03b2-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 \u03b2-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 \u03b2-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.<\/jats:p>","DOI":"10.1042\/bcj20160207","type":"journal-article","created":{"date-parts":[[2016,4,6]],"date-time":"2016-04-06T01:49:38Z","timestamp":1459907378000},"page":"1605-1615","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Role of post-translational modifications at the \u03b2-subunit ectodomain in complex association with a promiscuous plant P4-ATPase"],"prefix":"10.1042","volume":"473","author":[{"given":"Sara\u00a0R.","family":"Costa","sequence":"first","affiliation":[{"name":"Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Magdalena","family":"Marek","sequence":"additional","affiliation":[{"name":"Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristian\u00a0B.","family":"Axelsen","sequence":"additional","affiliation":[{"name":"Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark"},{"name":"SIB Swiss Institute of Bioinformatics, CMU, CH-1211, Geneva, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa","family":"Theorin","sequence":"additional","affiliation":[{"name":"Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas\u00a0G.","family":"Pomorski","sequence":"additional","affiliation":[{"name":"Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark"},{"name":"Faculty of Chemistry and Biochemistry, Department of Molecular Biochemistry, Ruhr University Bochum, Universit\u00e4tstrasse 150, D-44780 Bochum, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosa\u00a0L.","family":"L\u00f3pez-Marqu\u00e9s","sequence":"additional","affiliation":[{"name":"Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease-PUMPKin, University of Copenhagen, DK-1871, Frederiksberg C, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2016,5,27]]},"reference":[{"key":"2021112216290582800_B1","doi-asserted-by":"publisher","first-page":"243","DOI":"10.1146\/annurev.biophys.093008.131331","article-title":"P-Type ATPases","volume":"40","author":"Palmgren","year":"2011","journal-title":"Annu. Rev. Biophys."},{"key":"2021112216290582800_B2","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1007\/PL00006286","article-title":"Evolution of substrate specificities in the P-type ATPase superfamily","volume":"46","author":"Axelsen","year":"1998","journal-title":"J. Mol. Evol."},{"key":"2021112216290582800_B3","doi-asserted-by":"publisher","first-page":"1240","DOI":"10.1091\/mbc.E02-08-0501","article-title":"Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis","volume":"14","author":"Pomorski","year":"2003","journal-title":"Mol. Biol. Cell"},{"key":"2021112216290582800_B4","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1042\/BJ20120739","article-title":"Parkinson's disease-associated human P5B-ATPase ATP13A2 increases spermidine uptake","volume":"450","author":"De La Hera","year":"2013","journal-title":"Biochem. J."},{"key":"2021112216290582800_B5","doi-asserted-by":"publisher","first-page":"603","DOI":"10.1016\/j.bbalip.2009.02.005","article-title":"Mechanism and significance of P-4 ATPase-catalyzed lipid transport: lessons from a Na+\/K+-pump","volume":"1791","author":"Puts","year":"2009","journal-title":"Biochim. Biophys. Acta"},{"key":"2021112216290582800_B6","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1097\/MNH.0b013e3283036cbf","article-title":"Functional roles of Na,K-ATPase subunits","volume":"17","author":"Geering","year":"2008","journal-title":"Curr. Opin. Nephrol. Hypertens."},{"key":"2021112216290582800_B7","doi-asserted-by":"publisher","first-page":"40562","DOI":"10.1074\/jbc.M110.139543","article-title":"CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery","volume":"285","author":"Bryde","year":"2010","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B8","doi-asserted-by":"publisher","first-page":"268","DOI":"10.1002\/hep.21950","article-title":"ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity","volume":"47","author":"Paulusma","year":"2007","journal-title":"Hepatology"},{"key":"2021112216290582800_B9","doi-asserted-by":"publisher","first-page":"3418","DOI":"10.1091\/mbc.E03-11-0829","article-title":"Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae","volume":"15","author":"Saito","year":"2004","journal-title":"Mol. Biol. Cell"},{"key":"2021112216290582800_B10","doi-asserted-by":"publisher","first-page":"658","DOI":"10.1105\/tpc.107.054767","article-title":"The Arabidopsis P4-ATPase ALA3 localizes to the Golgi and requires a \u03b2-subunit to function in lipid translocation and secretory vesicle formation","volume":"20","author":"Poulsen","year":"2008","journal-title":"Plant Cell"},{"key":"2021112216290582800_B11","doi-asserted-by":"publisher","first-page":"23766","DOI":"10.1074\/jbc.M605214200","article-title":"Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3\u00a0in Leishmania parasites","volume":"281","author":"P\u00e9rez-Victoria","year":"2006","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B12","doi-asserted-by":"publisher","first-page":"43159","DOI":"10.1074\/jbc.M508262200","article-title":"Recombinant addition of N-glycosylation sites to the basolateral Na,K-ATPase \u03b21 subunit results in its clustering in caveolae and apical sorting in HGT-1 cells","volume":"280","author":"Vagin","year":"2005","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B13","doi-asserted-by":"publisher","first-page":"34794","DOI":"10.1074\/jbc.M302899200","article-title":"Mutational analysis of \u03b1\u2013\u03b2 subunit interactions in the delivery of Na,K-ATPase heterodimers to the plasma membrane","volume":"278","author":"Laughery","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B14","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1093\/oxfordjournals.jbchem.a124749","article-title":"Functional consequences of substitution of the disulfide-bonded segment, Cys127\u2013Cys150, located in the extracellular domain of the Na,K-ATPase \u03b2 subunit: Argl48 is essential for the functional expression of Na,K-ATPase","volume":"117","author":"Ueno","year":"1995","journal-title":"J. Biochem."},{"key":"2021112216290582800_B15","doi-asserted-by":"publisher","first-page":"6737","DOI":"10.1021\/bi00077a029","article-title":"An essential role for the extracellular domain of the Na,K-ATPase \u03b2-subunit in cation occlusion","volume":"32","author":"Lutsenko","year":"1993","journal-title":"Biochemistry"},{"key":"2021112216290582800_B16","doi-asserted-by":"publisher","first-page":"13249","DOI":"10.1074\/jbc.M111.313916","article-title":"Phosphatidylserine stimulation of Drs2p-Cdc50p lipid translocase dephosphorylation is controlled by phosphatidylinositol 4-phosphate","volume":"287","author":"Jacquot","year":"2012","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B17","doi-asserted-by":"publisher","first-page":"1227","DOI":"10.1007\/s00424-013-1363-4","article-title":"P4-ATPases: lipid flippases in cell membranes","volume":"466","author":"Lopez-Marques","year":"2014","journal-title":"Pfl\u00fcgers Arch."},{"key":"2021112216290582800_B18","doi-asserted-by":"publisher","first-page":"2708","DOI":"10.1016\/j.febslet.2010.04.071","article-title":"P4 ATPases: the physiological relevance of lipid flipping transporters","volume":"584","author":"Paulusma","year":"2010","journal-title":"FEBS Lett."},{"key":"2021112216290582800_B19","doi-asserted-by":"publisher","first-page":"323","DOI":"10.1016\/j.bbrc.2006.03.095","article-title":"Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Letn3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast","volume":"344","author":"Noji","year":"2006","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"2021112216290582800_B20","doi-asserted-by":"publisher","first-page":"40088","DOI":"10.1074\/jbc.M110.139006","article-title":"Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P-4-ATPases","volume":"285","author":"van der Velden","year":"2010","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B21","doi-asserted-by":"publisher","first-page":"17205","DOI":"10.1074\/jbc.M111.229419","article-title":"Critical role of the \u03b2-subunit CDC50A in the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2","volume":"286","author":"Coleman","year":"2011","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B22","doi-asserted-by":"publisher","first-page":"e33042","DOI":"10.1371\/journal.pone.0033042","article-title":"A putative plant aminophospholipid flippase, the Arabidopsis P4 ATPase ALA1, localizes to the plasma membrane following association with a \u03b2-subunit","volume":"7","author":"L\u00f3pez-Marqu\u00e9s","year":"2012","journal-title":"PLoS One"},{"key":"2021112216290582800_B23","doi-asserted-by":"publisher","first-page":"7649","DOI":"10.1038\/ncomms8649","article-title":"A phospholipid uptake system in the model plant Arabidopsis thaliana","volume":"6","author":"Poulsen","year":"2015","journal-title":"Nat. Commun."},{"key":"2021112216290582800_B24","doi-asserted-by":"publisher","first-page":"791","DOI":"10.1091\/mbc.E09-08-0656","article-title":"Intracellular targeting signals and lipid specificity determinants of the ALA\/ALIS P4-ATPase complex reside in the catalytic ALA \u03b1-subunit","volume":"21","author":"L\u00f3pez-Marqu\u00e9s","year":"2010","journal-title":"Mol. Biol. Cell"},{"key":"2021112216290582800_B25","doi-asserted-by":"publisher","first-page":"30529","DOI":"10.1074\/jbc.M112.371088","article-title":"Mapping functional interactions in a heterodimeric phospholipid pump","volume":"287","author":"Puts","year":"2012","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B26","doi-asserted-by":"publisher","first-page":"37855","DOI":"10.1074\/jbc.M205564200","article-title":"A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae","volume":"277","author":"Kato","year":"2002","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B27","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1042\/BJ20131318","article-title":"Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit","volume":"459","author":"Garc\u00eda-S\u00e1nchez","year":"2014","journal-title":"Biochem. J."},{"key":"2021112216290582800_B28","doi-asserted-by":"publisher","first-page":"3162","DOI":"10.1091\/mbc.E02-03-0172","article-title":"An essential subfamily of Drs2p-related P-type ATPases is required for protein trafficking between Golgi complex and endosomal\/vacuolar system","volume":"13","author":"Hua","year":"2002","journal-title":"Mol. Biol. Cell"},{"key":"2021112216290582800_B29","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1016\/S0076-6879(02)50957-5","article-title":"Transformation of yeast by lithium acetate\/single-stranded carrier DNA\/polyethylene glycol method","volume":"350","author":"Gietz","year":"2002","journal-title":"Methods Enzymol."},{"key":"2021112216290582800_B30","doi-asserted-by":"publisher","first-page":"953","DOI":"10.1007\/s11427-011-4230-6","article-title":"Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate","volume":"54","author":"Liu","year":"2011","journal-title":"Sci. China Life Sci."},{"key":"2021112216290582800_B31","doi-asserted-by":"publisher","first-page":"349","DOI":"10.1006\/meth.1999.0796","article-title":"Overexpression of multisubunit replication factors in yeast","volume":"18","author":"Burgers","year":"1999","journal-title":"Methods"},{"key":"2021112216290582800_B32","doi-asserted-by":"publisher","first-page":"462","DOI":"10.1104\/pp.103.027979","article-title":"A gateway cloning vector set for high-throughput functional analysis of genes in planta","volume":"133","author":"Curtis","year":"2003","journal-title":"Plant Physiol."},{"key":"2021112216290582800_B33","doi-asserted-by":"publisher","first-page":"616","DOI":"10.1111\/j.1365-313X.2005.02617.x","article-title":"Gateway-compatible vectors for plant functional genomics and proteomics","volume":"45","author":"Earley","year":"2006","journal-title":"Plant J."},{"key":"2021112216290582800_B34","doi-asserted-by":"crossref","first-page":"12341","DOI":"10.1016\/S0021-9258(19)49845-1","article-title":"Functional expression of plant plasma membrane H+-ATPase in yeast endoplasmic reticulum","volume":"267","author":"Villalba","year":"1992","journal-title":"J. Biol. Chem."},{"key":"2021112216290582800_B35","doi-asserted-by":"publisher","first-page":"383","DOI":"10.1007\/BF00331014","article-title":"The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type\u00a0of Agrobacterium binary vector","volume":"204","author":"Koncz","year":"1986","journal-title":"Mol. Gen. Genet."},{"key":"2021112216290582800_B36","doi-asserted-by":"publisher","first-page":"383","DOI":"10.1007\/978-1-4939-3179-8","article-title":"Transient expression of P-type ATPases in tobacco epidermal cells","volume":"1377","author":"Poulsen","year":"2016","journal-title":"Methods Mol. Biol."},{"key":"2021112216290582800_B37","doi-asserted-by":"publisher","first-page":"949","DOI":"10.1046\/j.1365-313X.2003.01676.x","article-title":"An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus","volume":"33","author":"Voinnet","year":"2003","journal-title":"Plant J."},{"key":"2021112216290582800_B38","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1006\/jmbi.2000.4042","article-title":"T-Coffee: a novel method for fast and accurate multiple sequence alignment","volume":"302","author":"Notredame","year":"2000","journal-title":"J. Mol. Biol."},{"key":"2021112216290582800_B39","doi-asserted-by":"publisher","first-page":"576","DOI":"10.1016\/j.sbi.2011.08.005","article-title":"Mechanisms and principles of N-linked protein glycosylation","volume":"21","author":"Schwarz","year":"2011","journal-title":"Curr. Opin. Struct. Biol."},{"key":"2021112216290582800_B40","doi-asserted-by":"publisher","first-page":"2027","DOI":"10.1016\/0006-2952(91)90604-4","article-title":"Mode of action of the lanthionine-containing peptide antibiotics duramycin, duramycin B and C, and cinnamycin as indirect inhibitors of phospholipase A2","volume":"42","author":"M\u00e4rki","year":"1991","journal-title":"Biochem. Pharmacol."},{"key":"2021112216290582800_B41","doi-asserted-by":"publisher","first-page":"611","DOI":"10.1016\/j.cell.2006.06.040","article-title":"Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast","volume":"126","author":"Parsons","year":"2006","journal-title":"Cell"},{"key":"2021112216290582800_B42","doi-asserted-by":"publisher","first-page":"e62577","DOI":"10.1371\/journal.pone.0062577","article-title":"Loss of the Arabidopsis thaliana P4-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative, pollen, and ovule development","volume":"8","author":"McDowell","year":"2013","journal-title":"PLoS One"}],"container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/473\/11\/1605\/685699\/bj4731605.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/473\/11\/1605\/685699\/bj4731605.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,22]],"date-time":"2021-11-22T23:15:25Z","timestamp":1637622925000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/473\/11\/1605\/49205\/Role-of-post-translational-modifications-at-the"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,5,27]]},"references-count":42,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2016,6,1]]}},"URL":"https:\/\/doi.org\/10.1042\/bcj20160207","relation":{},"ISSN":["0264-6021","1470-8728"],"issn-type":[{"value":"0264-6021","type":"print"},{"value":"1470-8728","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,5,27]]}}}