title: N-terminal proline-rich domain is required for scrambling activity of human phospholipid scramblases.
abstract: Human phospholipid scramblase 1 (hPLSCR1), a type II integral class membrane protein, is known to mediate bidirectional scrambling of phospholipids in a Ca(2+)-dependent manner. hPLSCR2, a homolog of hPLSCR1 that lacks N-terminal proline-rich domain (PRD), did not show scramblase activity. We attribute this absence of scramblase activity of hPLSCR2 to the lack of N-terminal PRD. Hence to investigate the above hypothesis, we added the PRD of hPLSCR1 to hPLSCR2 (PRD-hPLSCR2) and checked whether scramblase activity was restored. Functional assays showed that the addition of PRD to hPLSCR2 restored scrambling activity, and deletion of PRD in hPLSCR1 (ΔPRD-hPLSCR1) resulted in a lack of activity. These results suggest that PRD is crucial for the function of the protein. The effects of the PRD deletion in hPLSCR1 and the addition of PRD to hPLSCR2 were characterized using various spectroscopic techniques. Our results clearly showed that hPLSCR1 and PRD-hPLSCR2 showed Ca(2+)-dependent aggregation and scrambling activity, whereas hPLSCR2 and ΔPRD-hPLSCR1 did not show aggregation and activity. Thus we conclude that scramblases exhibit Ca(2+)-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.
authors.affiliation: From the Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600 036, India.
authors.forename: Sarika
authors.initials: S
authors.lastname: Rayala
authors.forename: Vincent G
authors.initials: VG
authors.lastname: Francis
authors.forename: Ulaganathan
authors.initials: U
authors.lastname: Sivagnanam
authors.forename: Sathyanarayana N
authors.initials: SN
authors.lastname: Gummadi
mesh_terms: D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008956:Models, Chemical; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: C493422:PLSCR1 protein, human; C493423:PLSCR2 protein, human; D049294:Phospholipid Transfer Proteins; D002118:Calcium
keywords: Calcium-binding Proteins; Fluorescence; Human Phospholipid Scramblase; Membrane Biophysics; Membrane Proteins; Membrane Reconstitution; Proline-rich Domain; Protein Aggregation