Osteoclasts are central regulators of skeletal bone mass and are the only cell type capable of digesting bone. To achieve this, osteoclasts have evolved specialized lysosome-related organelles (LROs) termed ‘secretory lysosomes’ that give rise to the ruffled border upon synchronous fusion with the bone-apposed plasma membrane. Fusion of secretory lysosomes with the ruffled border equips its membrane with sets of nanoscale machinery that are requisite for extracellular acidification and bone digestion. Yet, despite their obvious importance in osteoclast function and thus fertile ground for the discovery of new anti-resorptive drug targets, our understanding of the molecular anatomy of the osteoclast secretory lysosome remains limited. In particular, we still lack elementary information regarding the nature and number of membrane proteins that define secretory lysosome identity and function.
Here, to expand the molecular inventory of membrane proteins operating on osteoclast secretory lysosomes, we have combined biochemical organelle enrichment methods with high-resolution tandem mass spectrometry (nLC-ESI-MS/MS) to unbiasedly survey the osteoclast secretory lysosome membrane proteome.
Using this approach, we unambiguously identified 4153 unique proteins. Of these, 181 integral membrane proteins were functionally assigned as membrane transport proteins (transportome) and 390 as regulators of membrane trafficking (traffickome). Stratification of the ‘transportome’ and ‘traffickome’ for proteins that: (i) are significantly enriched on secretory lysosome membranes (LogFC >1.5 and p< 0.05) and (ii) whose corresponding human orthologue lead SNPs reached genome-wide significance (p< 5 x10-8) when cross-referenced against the largest genome-wide association study for a bone structural trait performed to date (eBMD, UKBioBank) established high-confidence lists of membrane transporters and trafficking proteins. By combining high-throughput siRNA-mediated gene knockdown studies in osteoclasts together with high-resolution confocal microscopy and novel genetic mouse models we demonstrate the power and utility of our approach to unmask new and physiologically relevant regulators of osteoclast function and skeletal bone mass.