Jos Jansen

6 137 Discussion and conclusion Furthermore, depletion of either protein resulted in decreased lysosomal protein degradation and decreased acidification of endosomal compartments. Although no direct evidence is available yet, it seems likely that human TMEM199 and CCDC115 are V0 assembly factors just as their yeast orthologs. Vph2p and Vma22p are ER-dedicated and have no known additional functions. However, it seems that TMEM199 and CCDC115 gained additional, yet uncharacterized, functions. Vph2p and Vma22p are located in the ER and not in COPI or COPII vesicles in yeast, in contrast to Vma21p which was demonstrated to shuttle between the ER and the Golgi. Epitope-tagged CCDC115 and TMEM199 were located outside the ER primarily in the ERGIC and the ER-to- Golgi transport vesicles. One possible explanation for this difference could be that V0 domain assembly takes place in the ERGIC. Another option is that assembly takes place in the ER and CCDC115 and TMEM199 chaperone the V0 domain via ERGIC vesicles to the Golgi. In this model, partial co-localization with COPII markers could indicate diffusion from the ER to the ERGIC, which is microtubuli independent. A lack of co-localization with ER-marker PDI could be due to unknown environmental or experimental factors. Complete or partial colocalization with COPI, COPII, Golgi and ERGIC markers predict that CCDC115 and TMEM199 travel with the V0 domain to the cis-Golgi, similar to Vma21p. However, both proteins lack a clear ER-retention motif. Co-localization with the V-ATPase was not possible due to lack of good-functioning antibodies. Non-canonical V-ATPase functions The V-ATPase is a highly conserved proton pump designed by Nature hundreds of millions years ago present in all eukaryotes.(13) The last decades additional functions besides acidification were discovered. It is a possibility that V-ATPase assembly factors play a direct or regulatory role in non-canonical functions of the V-ATPase. These included intracellular signaling through Wnt and Notch pathways, mTORC1 activation with subsequent cell growth and autophagy.(14- 16) It will be interesting to discover if and how CCDC115 and TMEM199 influence these non-canonical functions.