Expression of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes

Elisabeth Corvazier, Raymonde Bredoux, Tünde Kovács, Jocelyne Enouf

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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) Arg334, Arg396 and Arg638 were directly assigned to the ETF and ii) Arg198 was assigned as the only tryptic site to the LTF. Arg671, Lys712/Lys713 and Lys728 were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl2, EGTA or AlF4- 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) Arg1002 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.

Original languageEnglish
Pages (from-to)587-599
Number of pages13
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1788
Issue number3
DOIs
Publication statusPublished - Mar 1 2009

Keywords

  • Ca
  • HEK-293 cell
  • Isoform
  • Platelet
  • Proteolysis trypsin
  • SERCA3
  • Sarco/endoplasmic reticulum Ca ATPase
  • Site-directed mutagenesis
  • TG
  • tBHQ

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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