Expression of sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes

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) 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.