Processing of surfactant protein C requires a type II transmembrane topology directed by juxtamembrane positively charged residues.
Surfactant protein C (SP-C) is a lung-specific protein that is synthesized as a 21-kDa integral membrane propeptide (pro-SP-C) and proteolytically processed to a 3.7-kDa secretory product. Previous studies have shown that palmitoylation of pro-SP-C is dependent on two N-terminal juxtamembrane positively charged residues. We hypothesized that these residues influence modification of pro-SP-C by directing transmembrane orientation. Double substitution mutation of these juxtaposed residues from positive to neutral charged species resulted in complete reversal of transmembrane orientation of pro-SP-C and total abrogation of post-translational processing. Mutation of a single residue resulted in mixed orientation. Protein trafficking studies in A549 cells showed that while the double mutant was retained in the endoplasmic reticulum, single mutants produced a mixed pattern of both endoplasmic reticulum (double mutant-like) and vesicular (wild type-like) expression. Our study demonstrates the crucial role juxtamembrane positively charged residues play in establishing membrane topology and their influence on the trafficking and processing of pro-SP-C. Moreover this study provides a likely precedent for a mechanism in disorders associated with mutations in the membrane-flanking region of integral membrane proteins.
Mulugeta
Surafel
S
Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4318, USA. mulugeta@mail.med.upenn.edu
Beers
Michael F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D037721:Pulmonary Surfactant-Associated Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats
D018076:DNA, Complementary; D000939:Epitopes; D008164:Luminescent Proteins; D037721:Pulmonary Surfactant-Associated Protein C; D049452:Green Fluorescent Proteins; D001120:Arginine; D010450:Endopeptidases; D008239:Lysine