Expression of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes.
The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg(334), Arg(396) and Arg(638) were directly assigned to the ETF and ii) Arg(198) was assigned as the only tryptic site to the LTF. Arg(671), Lys(712)/Lys(713) and Lys(728) were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl(2), EGTA or AlF(4)(-) strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg(1002) as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.
Corvazier
Elisabeth
E
INSERM U 689, CRCIL, Hôpital Lariboisière, 41, Boulevard de la Chapelle, 75475 Paris Cedex 10, France.
Bredoux
Raymonde
R
Kovács
Tünde
T
Enouf
Jocelyne
J
D017607:Aluminum Compounds / Q000494:pharmacology; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D004721:Endoplasmic Reticulum; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D019284:Thapsigargin / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D017607:Aluminum Compounds; D006873:Hydroquinones; D007527:Isoenzymes; D010446:Peptide Fragments; C050992:tetrafluoroaluminate; D004533:Egtazic Acid; D019284:Thapsigargin; C018855:2-tert-butylhydroquinone; D014357:Trypsin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506949:ATP2A3 protein, human; D005459:Fluorides; D002118:Calcium