Hyperacidification of Trans‐Golgi Network and Endo/Lysosomes in Melanocytes by Glucosylceramide‐Dependent V‐ATPase Activity

Abstract

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H + -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K i for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.

A novel sphingolipid-TORC1 pathway critically promotes postembryonic development in Caenorhabditis elegans

“…By examining lysosomal markers LMP-1::GFP (Artal-Sanz et al, 2006; O’Rourke et al, 2009; Rabbitts et al, 2008), GLO-1::GFP (Schroeder et al, 2007; Zhang et al, 2010), and Neutral red (Long et al, 2002), we did not observe obvious defects in lysosome formation in elo-5(−) mutants (Figure 4—figure supplements 4 and 5). Major defects in lysosome formation would also be inconsistent with our genetic suppression data and previous studies on GlcCer by others (Entchev et al, 2008; van der Poel et al, 2011). One possible mechanism is that d17iso-GlcCer acts through the V-ATPase pathway, since the lysosomal V-ATPase has been shown to be stimulated by GlcCer and to play an essential role in TORC1 activation in studies using tissue culture cells (van der Poel et al, 2011; Zoncu et al, 2011; Bar-Peled et al, 2012).…”

“…Major defects in lysosome formation would also be inconsistent with our genetic suppression data and previous studies on GlcCer by others (Entchev et al, 2008; van der Poel et al, 2011). One possible mechanism is that d17iso-GlcCer acts through the V-ATPase pathway, since the lysosomal V-ATPase has been shown to be stimulated by GlcCer and to play an essential role in TORC1 activation in studies using tissue culture cells (van der Poel et al, 2011; Zoncu et al, 2011; Bar-Peled et al, 2012). Further biochemical and genetic analyses are needed to test this hypothesis in C. elegans .…”

Intracellular Proton Pumps as Targets in Chemotherapy: V-ATPases and Cancer

“…Other studies have corroborated genetically the importance of sphingolipids for V-ATPase function [83]. Moreover, luminal acidification of melanocyte trans-Golgi system and endo/lysosomes have been suggested to depend on glycosphingolipids, since a cell line devoid of glucosylceramide synthase presents defective luminal acidification [84]. Indeed, a regulatory function for lipid raft lipids in vivo can be envisaged: in a seminal study by Lafourcade et al [85] they observed that the V 1 /V 0 assembly ratio varies along the endocytic pathway in a way that correlates with the lipid-raft abundance and the luminal pH in these organelles.…”

Approaches for detecting lysosomal alkalinization and impaired degradation in fresh and cultured RPE cells: Evidence for a role in retinal degenerations

“…In Gaucher’s disease, the accumulation of cholesterol was associated with a secondary elevation in lysosomal pH (Sillence, 2013). Interestingly, reducing glucosylceramide synthase either chemically (Sillence, 2013) or molecularly (van der Poel et al, 2011) also alkalinized lysosomes, with the latter study suggesting glucosylceramide stimulates the vHATPase pump. This suggests that optimal levels of the protol pump require a balanced intermediate level of glucosylceramide.…”