Jos Jansen

78 Chapter 3 secretory pathway or the extracellular matrix in specialized cells. However, it has numerous additional functions and associations with other cellular mechanisms and pathways.(13) A few human disorders involving defects in V-ATPase proteins are known, including X-linked myopathy with excessive autophagy due to mutations in VMA21 (MIM: 310440),(23) renal tubular acidosis with deafness due to mutations in the kidney-specific isoforms ATP6V1B1 (MIM: 267300) or ATP6V0A4 (MIM: 602722),(24–26) Zimmerman- Laband syndrome 2 due to mutations in ATP6V1B2 (MIM: 616455),(27) osteopetrosis due to mutations in the osteoclast-specific V0a3 subunit (MIM: 259700) and cutis laxa due to mutations in ATP6V0A2.(24,28,29) ATP6V0A2 encodes the a2 subunit of the V0 domain, which localizes the V-ATPase complex to the Golgi apparatus, and is the only core subunit thus far associated with abnormal glycosylation.(11,30) The glycosylation profile seen in ATP6V0A2 deficiency is similar to that of TMEM199 deficiency; however, the clinical symptoms are clearly different. In addition, a clear effect of TMEM199 mutations was observed on organelle ultrastructure, different from ATP6V0A2 deficiency.(12) At the moment, it is difficult to explain the broad heterogeneity in clinical phenotypes among the different V-ATPase associated diseases. Even mutations in TMEM199 and CCDC115 , whose gene products are probably interacting in humans, result in different phenotypes. It seems plausible to attribute these differences to different functions of the V-ATPase. On the basis of our experiments, we propose a role for TMEM199 in Golgi homeostasis. However, it remains to be investigated whether this function is mediated through deficient proton transport via an effect on the V-ATPase or whether TMEM199 exerts additional roles in ER-to-Golgi transport. ► Figure 4. Localization of TMEM199 TMEM199 cDNA, compatible with Gateway Technology (Life Technologies), was purchased from Harvard Medical School. HeLa cells were transfected with FuGENE HD Transfection Reagent (Promega), grown on coverslips coated with poly-L-Lysine (Sigma), and incubated overnight. After permeabilization and fixation, cells were stained with fluorescently labeled antibodies against V5 (green in merge) and organelle markers (magenta in merge). Shown are representative cells for the V5 antibody, organelle markers, and a merge with DAPI stain (blue), including a 3-fold magnification. Co-localization is indicated by white in the merged channel. The graphs show the fluorescence intensity profiles along the cross-sections indicated. Scale bars represent 5 μ m. Organelle markers used are as follows: anti-V5-Tag (dilution 1:200, Life Technologies, #R960-25), anti-PDI (1:500, Abcam, #ab3672), anti-ERGIC-53 (1:200, Sigma-Aldrich, #E1031), anti- Giantin (1:1,000, BioLegend, #prb-114c), anti- BetaCOP (1:1,000, Abcam, #ab2899), and anti-Sec31a (1:500, Sigma-Aldrich, #HPA005457