Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization

Abstract

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 (t1/2 ≈ 50min) than that of the LPC polypeptide backbone (t1/2 ≈ 3h), 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.

Internalization of Proprotein Convertase PC7 from Plasma Membrane Is Mediated by a Novel Motif

“…This region of ϳ25 amino acids contains the two cysteines that are reversibly palmitoylated (10). However, these cysteines have been shown to affect the half-life of PC7 but not its TGN localization (11). Another remarkable feature is the presence of seven basic amino acids near these cysteines, of which five are clustered in the sequence HRSRKAK.…”

“…Overexpression studies have shown partial co-localization with TGN markers, but a less confined expression pattern (10,11). Ultrastructural analysis showed immunoreactivity associated with coated buds and vesicles adjacent to the Golgi stack.…”

“…Plasmids and Mutagenesis-The cloning of the 2.6-kb human PC7 cDNA in the mammalian expression vector pcDNA3 (Invitrogen), the introduction of the FLAG epitope (amino acids DYKDDDDK) between the pro-and catalytic domain, and the mutants f-PC7-⌬cyt (lacking the cytoplasmic tail; stop codon after Ser 689 ) and f-PC7-H 708 (stop codon after His 708 ) have been described previously (10,11). PC7 constructs carrying the FLAG epitope are indicated as f-PC7.…”

“…Its propeptide is removed autocatalytically. PC7 undergoes additional post-translational modifications (10), such as palmitoylation of 2 cysteines in the cytosolic tail (11), sulfation (12), and N-glycosylation (7). PC7 is widely expressed and has been demonstrated in early embryonic stages (8) as well as in several adult tissues (5,7).…”

The Proprotein Convertase PC7

“…PC7 also undergoes a number of post-translational modifications, including N-glycosylation (5) and cytosolic tail Cys-palmitoylation (8). However, the zymogen activation mechanism is still unknown, as this requires exit of the prosegment-PC7 complex from the ER and/or separation of the inhibitory prosegment from mature PC7.…”