cerulenin and Lymphoma

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t(1/2) approximately 50 min) than that of the LPC polypeptide backbone (t(1/2) approximately 3 h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.

Topics: Amino Acid Substitution; Brefeldin A; Cerulenin; Cycloheximide; Cysteine; Cytosol; Enzyme Stability; Exocytosis; Fluorescent Antibody Technique, Indirect; Half-Life; Lymphoma; Membrane Microdomains; Monensin; Mutation; Palmitic Acid; Protein Processing, Post-Translational; Protein Sorting Signals; Protein Structure, Tertiary; Protein Transport; Serine Endopeptidases; Subtilisins; trans-Golgi Network; Tunicamycin

The murine invariant chain (Ii) was found to incorporate radioactive palmitic acid. This binding of fatty acid inhibits the formation of interchain S-S bonds, probably because the cysteine residue in the transmembrane region of the Ii chain is palmitylated. The inhibition of fatty acylation by cerulenin blocks further posttranslational maturation of the invariant chain as shown by two-dimensional gel electrophoresis of Ii immunoprecipitates. In particular, the addition of sialic acid residues is blocked. Thus, it appears that fatty acylation is essential for carbohydrate processing of the Ii chain.

Topics: Acylation; Animals; Antibodies, Monoclonal; Antigens, Differentiation, B-Lymphocyte; Cerulenin; Cysteine; Disulfides; Histocompatibility Antigens Class II; Lymphoma; Mice; Mice, Inbred BALB C; N-Acetylneuraminic Acid; Neuraminidase; Palmitic Acid; Palmitic Acids; Plasmacytoma; Protein Binding; Sialic Acids; Tunicamycin