Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking*

Abstract

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.

Palmitoylation of claudins is required for efficient tight-junction localization

“…Although tetraspanins are sometimes associated with lipid rafts, the buoyant density of tetraspanins that could not be palmitoylated was no different to that of wild-type proteins, suggesting that targeting to detergent-resistant lipid rafts is not the main function of palmitoylation of these proteins (Stipp et al, 2003). Palmitoylation of carboxypeptidase D also has no effect on lipid-raft localization (Kalinina and Fricker, 2003). In the present study, we find that transfected wild-type claudin-14 is enriched in the fractions containing caveolin relative to the mutant protein.…”

“…1). By analogy to comparable sequences in the tetraspanins (Stipp et al, 2003) and some other integral membrane proteins (Bijlmakers and Marsh, 2003;Kalinina and Fricker, 2003), we reasoned that these cysteines might be modified by fatty-acid acylation with palmitic acid. To test this possibility, we first used [ 3 H]-palmitic acid to label endogenous claudins in MDCK cells.…”

“…In some (Melkonian et al, 1999) but not all (Yang et al, 2002;Kalinina and Fricker, 2003) cases, palmitoylation of proteins enhances their association with detergent-resistant lipid rafts. To test whether palmitoylation of claudin-14 influenced its partitioning into lipid rafts, we compared the relative amounts of wild-type and mutant claudin-14 in DRMs (Fig.…”

“…Palmitoylation has been reported to be a requirement for the stability of some (Percherancier et al, 2001) but not all (Kalinina and Fricker, 2003) membrane proteins. This finding, together with the localization of the mutant claudins to lysosomes, prompted us to determine the half-life of induced wild-type claudin-14 and the 4S mutant.…”

Conformational Defects Slow Golgi Exit, Block Oligomerization, and Reduce Raft Affinity of Caveolin-1 Mutant Proteins

“…Palmitoylation can target proteins to rafts and thus also might sort these proteins into cholesterol-rich Golgi domains in the Golgi. Palmitoylation also enhances Golgi transport of some transmembrane proteins (van't Hof and Crystal, 2002;Kalinina and Fricker, 2003), possibly through the same mechanism.…”