title: A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.
abstract: Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.
authors.affiliation: Molecular Biophysics Research Group of the Hungarian Academy of Sciences and Department of Biophysics, Semmelweis University, Budapest, Hungary.
authors.forename: Géza
authors.initials: G
authors.lastname: Antalffy
authors.affiliation: Molecular Biophysics Research Group of the Hungarian Academy of Sciences and Department of Biophysics, Semmelweis University, Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Pászty
authors.affiliation: Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Karolina
authors.initials: K
authors.lastname: Varga
authors.affiliation: Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Luca
authors.initials: L
authors.lastname: Hegedűs
authors.affiliation: Hungarian National Blood Transfusion Service, Budapest, Hungary; Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Enyedi
authors.affiliation: Hungarian National Blood Transfusion Service, Budapest, Hungary. Electronic address: padanyi@biomembrane.hu.
authors.forename: Rita
authors.initials: R
authors.lastname: Padányi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002451:Cell Compartmentation; D002452:Cell Count; D002462:Cell Membrane / Q000201:enzymology*; D004285:Dogs; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D021382:Protein Sorting Signals; D049452:Green Fluorescent Proteins; D001216:Asparagine; C507517:ATP2B4 protein, human; D053499:Plasma Membrane Calcium-Transporting ATPases; D034281:Dynamins; D007930:Leucine; D008239:Lysine; D002118:Calcium
keywords: C-terminal tail; Di-leucine motif; EEA1; ER; Endocytosis; MDCK; Madin Darby canine kidney; NHE; Na(+)/H(+) exchanger; PDZ; PM; PMCA; PMCA4b; PSD-95 Dlg ZO-1; Plasma membrane expression; SERCA; WGA; early endosome antigen 1; endoplasmic reticulum; plasma membrane; plasma membrane Ca(2+) ATPase; sarco/endoplasmic reticulum Ca(2+) ATPase; wheat germ agglutinin