Am. J. Respir. Cell Mol. Biol.,
Volume 25, Number 2, August, 2001 156-163
The Juxtamembrane Lysine and Arginine Residues of Surfactant Protein C
Precursor Influence Palmitoylation via Effects on Trafficking
Anja
ten Brinke,
Joseph J.
Batenburg,
Barend M.
Gadella,
Henk P.
Haagsman,
Arie B.
Vaandrager,
and
Lambert M. G.
van Golde
Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, and Institute of Biomembranes and Department of the
Science of Food of Animal Origin, Faculty of Veterinary Medicine, Utrecht University, The Netherlands
Surfactant protein (SP)-C propeptide (proSP-C) becomes palmitoylated on cysteines 5 and 6 before mature SP-C is formed
by several proteolytic steps. To study the structural requirements for the palmitoylation of proSP-C, his-tagged human
proSP-C (his-proSP-C) and his-proSP-C mutants were expressed
in Chinese hamster ovary cells and analyzed by metabolic labeling with [3H]palmitate and immunocytochemistry. Substitution of cysteines 5 and 6 by serines showed that these were
the only two cysteine residues palmitoylated in his-proSP-C.
Substitution of the juxtamembrane basic residues lysine and
arginine by uncharged glutamines led to a large decrease in
palmitoylation level of proSP-C. The addition of brefeldin A
nearly abolished this decrease for the lysine and double mutant; the palmitoylation of the arginine mutant increased also,
but not to wild-type (WT) levels. Fluorescence immunocytochemistry showed that WT proSP-C was localized in punctate vesicles throughout the cell, whereas the mutant lacking
the juxtamembrane positive charges was found more perinuclear, probably in the endoplasmic reticulum (ER). This indicates that the two basic juxtamembrane residues influence
palmitoylation of proSP-C by preventing the transport of
proSP-C out of the ER, implying that proSP-C becomes palmitoylated normally in a compartment distal to the ER.
Abbreviations: brefeldin A, BFA; complementary DNA, cDNA; Chinese
hamster ovary, CHO; endoplasmic reticulum, ER; immunoglobulin, Ig;
phosphate-buffered saline, PBS; SP-C propeptide, proSP-C; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; surfactant
protein, SP; wild-type, WT.
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Copyright © 2001 American Thoracic Society.
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