DRP-1b - A Novel ZIPk-Like Isoform Figure 2. The DRP-1b alternative exon shows similarity to the extra-catalytic domain of ZIPk. A multiple sequence alignments of the extra catalytic domain of ZIPk and DRP-1b orthologs from the indicated vertebrates. Blue arrows- ZIPk autophosphorylation sites; red arrows- ZIPk phosphorylation sites by DAPk. Brown to yellow bars- conservation measure; the position of ZIPk leucine zipper is marked by pale blue boxes. The MSA was performed using CLUSTALW program and visualized with the JalView tool. doi:10.1371/journal.pone.0017344.g002 reported to date (Table S1), suggesting that unlike the ubiquitous expression of the three well known family members the new alternate isoform may have a more restricted pattern of expression. Experimentally, we identified the presence of DRP-1b mRNA in cDNA libraries of mouse embryos. The detection was done by PCR amplification, using specific probes on both sides of the alternative splice junction. DRP-1b mRNA was clearly detected in samples from embryonic days 10, 14, 16 and 18 (Figure 3A), proving the presence of this alternatively spliced mRNA in embryonic cells of different developmental stages (Note that the apparent lack of detection of DRP-1b mRNA in samples from day 12 is due to the fact that the quality of the sample is lower, as shown by the attenuated detection of DRP-1 mRNA, used as a control (Figure 3A)). We next searched for DRP-1b protein expression, using antibodies directed against the N’ terminus of DRP-1 (which is present in both isoforms), and which recognize both human and mouse proteins. The isoform distinction is done through the size of the protein, where DRP-1 runs on gels as a 42 kD band [13] and DRP-1b is predicted to display a size of 55 kD. Initial screen of various human and mouse cells lines, including HEK293T, HeLa, H1299, and NIH3T3, failed to detect a band of the appropriate size. We next screened brain extracts from fetal and young mice, and found a strong signal at the expected size, that was absent in adult mouse brains, suggesting strong protein expression of the DRP-1b isoform in the brains of embryos and young mice (Figure 3B). DRP-1 on the other hand was expressed in all the brain samples taken. DRP-1b isoform was also detected in human embryonic stem cells (Figure 3B). Thus, we proved that the DRP-1 locus undergoes alternative splicing in some tissues/cells from early developmental stages, and that the alternative transcript is translated into protein, both in mice and humans. Functional characterization of DRP-1b protein To characterize the properties of the DRP-1b protein we cloned FLAG-tagged DRP-1b in a mammalian expression vector. Over- expression of the protein in 293T cells led to extensive membrane blebbing (Figure 4A), at levels comparable to those induced by over-expression of DRP-1 and ZIPk (Figure 4, B and C). PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e17344