Phospholipid scramblases and Tubby-like proteins belong to a new superfamily of membrane tethered transcription factors

Abstract

Motivation: Phospholipid scramblases (PLSCRs) constitute a family of cytoplasmic membrane-associated proteins that were identified based upon their capacity to mediate a Ca(2+)-dependent bidirectional movement of phospholipids across membrane bilayers, thereby collapsing the normally asymmetric distribution of such lipids in cell membranes. The exact function and mechanism(s) of these proteins nevertheless remains obscure: data from several laboratories now suggest that in addition to their putative role in mediating transbilayer flip/flop of membrane lipids, the PLSCRs may also function to regulate diverse processes including signaling, apoptosis, cell proliferation and transcription. A major impediment to deducing the molecular details underlying the seemingly disparate biology of these proteins is the current absence of any representative molecular structures to provide guidance to the experimental investigation of their function.

Results: Here, we show that the enigmatic PLSCR family of proteins is directly related to another family of cellular proteins with a known structure. The Arabidopsis protein At5g01750 from the DUF567 family was solved by X-ray crystallography and provides the first structural model for this family. This model identifies that the presumed C-terminal transmembrane helix is buried within the core of the PLSCR structure, suggesting that palmitoylation may represent the principal membrane anchorage for these proteins. The fold of the PLSCR family is also shared by Tubby-like proteins. A search of the PDB with the HHpred server suggests a common evolutionary ancestry. Common functional features also suggest that tubby and PLSCR share a functional origin as membrane tethered transcription factors with capacity to modulate phosphoinositide-based signaling.

Fig. 1.

Multiple alignment showing members of the scramblase and DUF567 families (upper block) and the tubby-like family (lower block). Members within each family were aligned with the multiple alignment program PROMALS (Pei and Grishin, 2007). The scramblase and DUF567 sequences were aligned with each other using HHpred (Soding, 2005) in local MAC alignment mode (Soding et al., 2005), while keeping the family alignments frozen. The resulting alignment was merged with the alignment of tubby-like proteins by using the structural alignment of 1zxu and 1c8z from TMalign (Zhang et al., 2005) as a guide and again keeping sub-alignments frozen. Red boxes represent regions that are part of the structural alignment. Alignment columns for which scramblase and tubby family members exhibit similar amino acids are colored according to the chemical nature of the residue class. Various sequence features of the PLSCR1 sequence are indicated by colored, bold letters: transcriptional activation domain (magenta), Cysteine palmitoylation motif (orange), non-classical nuclear localization signal (green), Ca2⁺-binding motif (blue), predicted transmembrane helix (red).

Fig. 2.

3D structural model of PLSCR1 computed by homology modeling. PLSCR1 forms a closed, symmetric β -barrel of 12 β-strands wrapped around a very hydrophobic C-terminal helix. Various sequence features of PLSCR1 are highlighted in color: transcriptional activation domain (magenta), Cysteine palmitoylation motif (orange), non-classical nuclear localization signal (green), Ca²⁺-binding motif (blue), predicted transmembrane helix (red).