Two tail-anchored protein variants, differing in transmembrane domain length and intracellular sorting, interact differently with lipids.
C-tail-anchored (TA) proteins often require a transmembrane domain of moderate hydrophobicity to maintain their endoplasmic reticulum residence, but the suggested role of protein-lipid interactions in this phenomenon has not been established. Here, we studied the interaction of TA proteins with lipids by differential scanning calorimetry by using a model system consisting of liposomes embedding either of two forms of cytochrome b(5): the endoplasmic reticulum-resident wild-type (b(5)wt) and a mutant thereof (b(5)ext), that contains five extra nonpolar amino acids in its transmembrane domain and, therefore, reaches the plasma membrane. The proteins were incorporated into liposomes of palmitoyl-oleyl-phosphatidylcholine (POPC) or POPC mixed with either distearoyl-phosphatidylserine (DSPS), palmitoyl-oleyl-phosphatidylserine (POPS), distearoyl-phosphatidylcholine (DSPC), or C16-ceramide (CER). POPC liposomes displayed a single thermotropic transition centered at -3.4 degrees C. When present, the second lipid formed a domain within the POPC bilayer, as indicated by the appearance of an additional peak. This peak was centered at temperatures close to 0 degrees C in the case of liposomes containing 10% CER, DSPS, and POPS and at 23 degrees C in the case of DSPC, likely reflecting a higher degree of molecular packing for DSPC domains. In DSPS/POPC, POPS/POPC, or CER/POPC, but not in DSPC/POPC liposomes, the insertion of b(5)wt increased, whereas b(5)ext decreased, the relative contribution to the total enthalpy of the higher temperature, phase-separated component. These results were confirmed with fluorescence measurements by using pyrene-labeled phospholipids. The dissimilar interaction with lipids of these two differently localized TA proteins could have implications for their intracellular sorting.
Ceppi
Paolo
P
Department of Experimental, Environmental Medicine, and Biotechnologies (DIMESAB), Medical School, University of Milano-Bicocca, 20052 Monza, Italy.
Colombo
Sara
S
Francolini
Maura
M
Raimondo
Francesca
F
Borgese
Nica
N
Masserini
Massimo
M
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002462:Cell Membrane / Q000737:chemistry*; D015786:Cytochromes b5 / Q000737:chemistry*; D005453:Fluorescence; D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines; D017434:Protein Structure, Tertiary
D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes; D010713:Phosphatidylcholines; D015786:Cytochromes b5; C028694:1-palmitoyl-2-oleoylphosphatidylcholine