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

Abstract

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin ␣/␤.Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257 GKISKH-WTGI 266 . This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 Å crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin ␣. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin ␣, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin ␣.Nuclear transport is an active signal-mediated process that requires, in most cases, soluble transport factors and specific import signals. Two families of transport receptors have been identified in the importin ␤ superfamily, which is involved in both nuclear import and export, and the TAP superfamily that mediates nuclear export. Transport receptors recognize specific nuclear localization signals (NLSs) 1 and nuclear export signals exposed on the molecular surface of cargoes. In the classical nuclear import pathway proteins bearing a classical SV40-like NLS (PKKKRKV) are recognized by the importin ␣/␤ heterodimer (also known as karyopherin ␣/␤) (1-3). Importin ␣ (4) acts as an adaptor that recognizes NLS sequences after association with the receptor importin ␤ (5). The importin ␣/␤-NLS cargo complex is then translocated through the nuclear pore complex in a process that requires multiple rounds of interaction of the receptor importin ␤ with nucleoporins, likely via their exposed hydrophobic FG-rich motifs (6, 7

Antileukemic roles of human phospholipid scramblase 1 gene, evidence from inducible PLSCR1-expressing leukemic cells

“…Biochemically, PLSCR1 was proposed to be a lipid-raft-associated endofacial plasma membrane protein, which is phosphorylated by selective protein kinases such as c-Abl, c-Src and PKCd that participate in multiple growth factor receptor signaling pathways (Frasch et al, 2000;Sun et al, 2001Sun et al, , 2002Nanjundan et al, 2003). More recently, the nonpalmitoylated PLSCR1 was found to traffic into the nuclei via the non-classical nuclear localization signal (Ben-Efraim et al, 2004;Chen et al, 2005). In the nucleus, PLSCR1 tightly binds to sequence GTAAC CATGTGGA in the promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene to enhance expression of the receptor .…”

“…Of course, we can not completely exclude a possible contribution of the nuclear PLSCR1 to these events. To resolve this question, the effects of the palmitoylated site-mutated PLSCR1 (only expression in nuclei) and nuclear localization signal-mutated PLSCR1 (only expression in cytosol) (Chen et al, 2005) on leukemic cells deserve to be further observed in the future.…”

Nuclear Shuttling of She2p Couples ASH1 mRNA Localization to its Translational Repression by Recruiting Loc1p and Puf6p

“…Interestingly, its sequence is very divergent from classical monopartite and bipartite NLS because it contains only one lysine and is rich in hydrophobic residues. To our knowledge, all the currently reported NLS that bind directly importin ␣ contain at least two essential basic amino acids (Chen et al, 2005;Lange et al, 2007), suggesting that the repertoire of nuclear localization signals may be larger than suggested. Mutation of five conserved residues in this NLS disrupted the nuclear targeting of She2p and its interaction with Srp1p, confirming its role as a nuclear localization sequence.…”

A 24-residue presequence localizes human factor B to mitochondria

“…It is therefore plausible that the nuclear localization of a 20.3 kDa mature factor B could be achieved by its diffusion from the cytoplasm without employing a mechanism that relies on a specific recognition by importin α of the NLS motif within its potential cargo [17]. Indeed, we could not detect a canonical NSL motif Pro-Lys-Lys-Lys-Arg-Lys-Val within the amino acid sequence of factor B, which does not preclude the existence of a non-canonical NSL motif [19]. Surprisingly, we detected a short stretch comprised of Phe 161 -Lys-Thr-AlaLeu-Pro-Ser-Leu-Glu-Leu 170 amino acids in the C-terminus of the human factor B polypeptide that conforms to a leucine-rich nuclear export signal (NES) consensus motif Leu-x(2, 3)-[Leu,Ile,Val,Phe,Met]-x(2, 3)-Leu-x-[Leu,Ile] [20].…”