N-terminal proline rich domain is required for scrambling activity of human phospholipid scramblases [Molecular Biophysics]

March 19th, 2014 by Rayala, S., Francis, V. G., Sivagnanam, U., Gummadi, S. N.

Human phospholipid scramblase 1 (hPLSCR1), a type II integral class membrane protein known to mediate bi-directional scrambling of phospholipids (PLs) in a Ca2+dependent manner. hPLSCR2 a homolog of hPLSCR1, which lacks N-terminal proline rich domain (PRD) did not show scramblase activity. We attribute this absence of scramblase activity of hPLSCR2 due to the lack of N-terminal PRD. Hence to investigate the above hypothesis, we added the PRD domain of hPLSCR1 to hPLSCR2 (PRD-hPLSCR2) and checked whether scramblase activity was restored. Functional assays showed that addition of PRD domain to hPLSCR2 restored scrambling activity and deletion of PRD domain in hPLSCR1 (∆PRD-hPLSCR1) resulted in lack of activity. These results suggest that PRD domain is crucial for the function of the protein. The effect of PRD deletion in hPLSCR1 and addition of PRD to hPLSCR2 was characterized using various spectroscopic techniques. Our results clearly showed that hPLSCR1, and PRD-hPLSCR2 showed Ca2+ dependent aggregation and scrambling activity; whereas hPLSCR2 and ∆PRD-hPLSCR1 did not show aggregation and activity. Thus we conclude that scramblases exhibit Ca2+ dependent scrambling activity by aggregation of protein. Our results provide a possible mechanism for phospholipid scrambling mediated by PLSCRs and the importance of PRD in its function and cellular localization.