Jos Jansen

44 Chapter 2 To investigate whether we could rescue the phenotype of individual F1-II4, we transfected skin fibroblasts with a construct containing either a mock construct or CCDC115 wild-type sequence. As seen in Figures 3C and 3D, fibroblasts transfected with a mock construct have a non-detectable fluorescence intensity in the Golgi, in contrast to fibroblasts transfected with a construct containing wild-type CCDC115 . In addition to metabolic labeling, we stained healthy control fibroblasts and fibroblasts from affected individual F2-II1 with anti-calnexin antibody to visualize the ER (Figure 3E). Individual F2-II1 fibroblasts showed a dilated ER in 60% of counted cells, in comparison to 20% in healthy control fibroblasts. Localization To define the subcellular location of CCDC115, we constructed a pcDNA3.1- CCDC115 -V5 plasmid for transient expression of C-terminally V5-tagged CCDC115 in HeLa cells. Confocal imaging revealed clear localization to the ER-Golgi intermediate compartment (ERGIC) and coat protein complex I (COPI) vesicles (Figure 4). Also, in immortalized human hepatocytes (HepaRG), CCDC115 located to the ERGIC and COPI vesicles (data not shown). Partial co-localization was seen in both cell types with COPII and Golgi markers. No co-localization was seen with the ER marker PDI (Figure 4). We conclude that CCDC115 predominantly localizes to the ER-to-Golgi region but not to the ER, in contrast to yeast Vma22p.