Abstract
Carboxypeptidase D (CPD) functions in the processing of proteins that transit the secretory pathway, and is present in all vertebrates examined as well as Drosophila. Several forms of CPD mRNA were previously found in Drosophila that resulted from differential splicing of the gene. In the present study, Northern blot, reverse transcriptase PCR, and Western blot analysis showed that each splice variant occurs in a single cell type, the Drosophila-derived Schneider 2 (S2) cell line. The short forms containing a single carboxypeptidase domain were secreted from the S2 cells while the long forms containing three carboxypeptidase domains, a transmembrane domain, and one of two different cytosolic tails were retained in the cell. To investigate the role of the two different C-terminal tail sequences (tail-1 and tail-2) that result from the differential splicing within exon 8, constructs containing a reporter protein (albumin) attached to the transmembrane domain and tail-1 or tail-2 of CPD were expressed in S2 cells and a mouse pituitary cell line (AtT20 cells). Immunofluorescence analysis revealed different intracellular distributions of the two constructs, with the tail-2 construct showing considerable overlap with a Golgi marker. The two C-terminal tail sequences also resulted in different internalization efficiencies from the cell surface in both cell lines. Interestingly, the distribution and routing of the tail-2 form of Drosophila CPD in the AtT20 cells are similar to the previously characterized endogenous mouse CPD protein, indicating that the elements for this trafficking have been conserved between Drosophila and mammals.
