Converting a marginally hydrophobic soluble protein into a membrane protein.
δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.
Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, SE-106 91 Stockholm, Sweden.
Morten H H
MH
Nørholm
Fiona
F
Cunningham
Charles M
CM
Deber
Gunnar
G
von Heijne
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004285:Dogs; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D017433:Protein Structure, Secondary; D021381:Protein Transport; D000069816:SEC Translocation Channels
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D000069816:SEC Translocation Channels; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases