Regulation of organelle pH by glycosphingolipids

Even though only about 5 mol% glycosphingolipids are found in cellular membranes, glycosphingolipids are essential during development, as a lack of these lipids was embryonically lethal in mice. A mutant cell line GM95 deficient in these lipids, was derived from the pigmented mouse melanoma cells MEB4. The first observation was that pigmentation is lost in the mutant glycosphingolipid-deficient GM95 cells. This was caused by mislocalisation of tyrosinase, the rate-limiting enzyme in pigment synthesis. We were able to locate the sorting signal in the lumenal domain of melanocytic proteins. Subsequently, we found that a lumenal determinant, pH, was less acidic in the TGN and lysosomes of mutant GM95 cells compared to the wild-type MEB4 cell line. Interestingly, the pH in both organelles of GM95 was comparable to HeLa cells and mouse fibroblasts. Therefore, it is the wild-type MEB4 cell line that has an abnormally low pH in its TGN and lysosomes. A similar low pH was found in a different melanocyte cell line, melan-a. As far as we know, this is the first study reporting that melanocytes have an exceptionally low pH in the TGN and lysosomes. The pH in GM95 organelles was lowered to the level of wild-type cells after restoration of the glycosphingolipid content by transfection with the missing glucosylceramide synthase (GCS). In this thesis, we addressed the question how glycosphingolipids regulate the pH. In the cell line lacking glycosphingolipids, we found that two determinants of lumenal pH were affected: (1) We stumbled upon a novel GCS-dependent pathway for the expression of the putative anion channel Oca2. (2) In addition, the activity of the vacuolar-type H+-translocating ATPase (V-ATPase) was 2-3 fold reduced in the GM95 cells. Competition studies and labelling with photoactivatable and clickable GlcCer analogues indicate that the V-ATPase may be regulated via a direct interaction with glucosylceramide (GlcCer), the precursor of the higher glycosphingolipids. Glycosphingolipids have been implicated in functions as the impermeability barrier of the plasma membrane, cell-cell interactions and signalling. The identification of two additional functions of glycosphingolipids, regulation of gene expression and pH, assigns to the glycosphingolipids a versatile and central role in cell physiology