The juxtamembrane lysine and arginine residues of surfactant protein C precursor influence palmitoylation via effects on trafficking

Surfactant protein (SP)-C propeptide (proSP-C) becomes palmitoylated on cys teines 5 and 6 before mature SP-C is formed by several proteolytic steps. T o 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 i n Chinese hamster ovary cells and analyzed by metabolic labeling with [H-3] palmitate and immunocytochemistry. Substitution of cysteines 5 and 6 by ser ines showed that these were the only two cysteine residues palmitoylated in his-proSP-C. Substitution of the juxtamembrane basic residues lysine and a rginine by uncharged glutamines led to a large decrease in palmitoylation l evel of proSP-C. The addition of brefeldin A nearly abolished this decrease for the lysine and double mutant; the palmitoylation of the arginine mutan t increased also, but not to wild-type (WT) levels. Fluorescence immunocyto chemistry showed that WT proSP-C was localized in punctate vesicles through out the cell, whereas the mutant lacking the juxtamembrane positive charges was found more perinuclear, probably in the endoplasmic reticulum (ER). Th is indicates that the two basic juxtamembrane residues influence palmitoyla tion of proSP-C by preventing the transport of proSP-C out of the ER, imply ing that proSP-C becomes palmitoylated normally in a compartment distal to the ER.