Phospholipid Scramblase 1 Is Imported into the Nucleus by a Receptor-Mediated Pathway and Interacts with DNA^,

Abstract

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, Ca(2+)-binding, endofacial plasma membrane protein originally identified by its capacity to accelerate transbilayer movement of membrane phospholipids. We recently reported that when palmitoylation of PLSCR1 does not occur, it is localized to the nucleus rather than the plasma membrane. Nuclear localization of PLSCR1 was also observed upon induction of its de novo synthesis by cytokines such as interferon alpha that activate the PLSCR1 gene. Despite its capacity to enter the nucleus, its sequence does not predict a nuclear localization signal. To gain insight into the mechanism and potential significance of nuclear PLSCR1, we investigated the conditions required for its import and retention in the nucleus. We show that nuclear localization of PLSCR1 is dependent on cytosolic factors and energy. Furthermore, we show that PLSCR1 is specifically transported into the nucleus by the importin alpha/beta import pathway, and binds directly and with high affinity to importin alpha. Analysis of deletion mutants suggested that the NLS of PLSCR1 is between residues 242 and 290 and, furthermore, that a peptide within this region encompassing residues (257)GKISKHWTGI(266) is sufficient for nuclear import when conjugated to BSA. In addition, in intact cells, mutation of positively charged amino acids within this putative NLS in the full-length protein completely blocked its entry into the nucleus, consistent with its role in targeting PLSCR1 to the nucleus. Release of PLSCR1 from the nucleus was only observed after treatment of cells with both detergent and an elevated NaCl concentration, or following DNase treatment of the nucleus, suggesting ionic interactions of PLSCR1 with a nuclear component bound to genomic DNA or directly with genomic DNA. Purified PLSCR1 was also found to bind directly to a genomic DNA-cellulose conjugate, and its elution from DNA also required an elevated NaCl concentration. These data support a mechanism of receptor-mediated nuclear import of PLSCR1 and suggest a potential nuclear function for this plasma membrane protein.

Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator To Externalize Phosphatidylserine

“…Lastly, the nuclear localization of PLSCR1 in N1511 cells by day 7 of BMP/Ins treatment is similar to that observed in fibroblasts and suggests that PLSCR1 becomes depalmitoylated (67). The precise site of nuclear interaction is presently under investigation.…”

“…Interestingly, by day 10, the effect of cholesterol depletion (i.e., membrane raft disruption) on scramblase activity was almost negligible, most likely due to the redistribution of PLSCR1 to the nucleus ( Figure 3B, as seen previously (67). Collectively, these results suggest that PS externalization is dependent on the relative activities of scramblase and ALPT, most likely in a cholesterol-dependent manner.…”

Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

“…ResiduesBen-Efraim et al (30) showed that PLSCR1 is imported into the nucleus by the importin ␣/␤ heterodimer (30) and the NLS sequence responsible for nuclear import comprises residues 257 GKISKHWTGI 266 . This sequence is less basic than classical SV40 NLS (the calculated isoelectric point is ϳ9.8 versus 11.38 for the SV40 NLS) and significantly more hydrophobic.…”

“…In the cytoplasm PLSCR1 is localized mostly at the endofacial plasma membrane via multiple palmitoylations, where it binds calcium via a direct EF hand-like calcium binding domain (29). It was reported recently that PLSCR1 has the potential to enter the nucleus (30). This nuclear activity was observed either in the presence of inhibitors of palmitoyl-CoA, which block PLSCR1 anchoring to the plasma membrane, or by treatment with interferon ␣ (31), which is known to activate the PLSCR1 gene expression transcriptionally (32,33).…”

“…In the nucleus PLSCR1 might function in the overall cellular response of those cytokines and growth factors that can induce expression of the PLSCR1 gene. The PLSCR1 NLS was mapped to the C terminus region 257 GKISKHWTGI 266 , and it was reported to be sufficient for nuclear import when conjugated to bovine serum albumin via association with the importin ␣/␤ heterodimer (30).…”