Membrane binding and insertion of the predicted transmembrane domain of human scramblase 1

Abstract

Human phospholipid scramblase 1 (SCR) was originally described as an intrinsic membrane protein catalyzing transbilayer phospholipid transfer in the absence of ATP. More recently, a role as a nuclear transcription factor has been proposed for SCR, either in addition or alternatively to its capacity to facilitate phospholipid flip-flop. Uncertainties exist as well from the structural point of view. A predicted α-helix (aa residues 288-306) located near the C-terminus has been alternatively proposed as a transmembrane domain, or as a protein core structural element. This paper explores the possibilities of the above helical segment as a transmembrane domain. To this aim two peptides were synthesized, one corresponding to the 19 α-helical residues, and one containing both the helix and the subsequent 12-residues constituting the C-end of the protein. The interaction of these peptides with lipid monolayers and bilayers was tested with Langmuir balance surface pressure measurements, proteoliposome reconstitution and analysis, differential scanning calorimetry, tests of bilayer permeability, and fluorescence confocal microscopy. Bilayers of 28 different lipid compositions were examined in which lipid electric charge, bilayer fluidity and lateral heterogeneity (domain formation) were varied. All the results concur in supporting the idea that the 288-306 peptide of SCR becomes membrane inserted in the presence of lipid bilayers. Thus, the data are in agreement with the possibility of SCR as an integral membrane protein, without rejecting alternative cell locations.

A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

“…The behavior of short and long peptides with other lipid compositions, such as pure pSM and pure DPPC, was checked previously (13) and the obtained endotherms were virtually the same for both peptides. Thus, the endothermic differences obtained with SOPC/Chol membranes in this case are apparently the result of the lack of the Cter extracellular coil in TM19 peptide, suggesting that this segment is a crucial element for the interaction with Chol.…”

“…Although mutant SCR lacking the TMD is still able to bind lipid bilayers (10), it has lost functional scramblase activity (11,12). Moreover, the isolated TMD binds and becomes inserted into lipid bilayers with great affinity (13).…”

Comparative genomic analyses of transport proteins encoded within the red algae Chondrus crispus, Galdieria sulphuraria, and Cyanidioschyzon merolae11

“…), the Outer Membrane Mitochondrial Cholesterol/Porphyrin Uptake Translocator Protein (TSPO) Family (TC# 9.A.24; Batarseh and Papadopoulos ), of which C. merolae also has one member, the Ca 2+ ‐dependent Phospholipid Scramblase Family (TC# 9.A.36; Posada et al. ), and the aforementioned VAP proteins in the ER that function in vesicle biogenesis, exocytosis, and protein stabilization (Lev et al. ).…”

Topological characterization of the mitochondrial phospholipid scramblase 3

“…Several studies suggest the scramblase subfamily domain is a true transmembrane helix . This α‐helix has been designated as responsible for anchoring the protein to the plasma or mitochondrial membranes.…”