Oral Presentation 29th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2019

Deciphering the Secretory Lysosome ‘Transportome’ Unveils Novel Regulators of Osteoclast Function and Bone Homeostasis (#17)

Amy B P Ribet 1 , Daniel Yagoub 1 , Laila N Abudulai 1 , Pei Ying Ng 1 , Nathan J Pavlos 1
  1. UWA, CRAWLEY, WA, Australia

 

The ruffled border is a lysosome-related bone-digesting organelle unique to osteoclasts. It is formed upon the fusion of secretory lysosomes with the bone-apposed plasma membrane. Fusion of secretory lysosomes with the ruffled border releases cathepsin k into the underlying resorptive space. At the same time, it equips the ruffled border with sets of lysosomal membrane proteins that are requisite for bone resorptive function. Despite its crucial importance, our understanding of the ruffled border’s molecular anatomy and its secretory lysosomal progenitors remains limited. In particular, we still lack elementary information on the protein composition of the ruffled border, including the numbers and identities of lysosomal membrane residents whose usual functions are to facilitate the exchange of molecules across its membrane (i.e. transporters). To extend the molecular inventory of transporters operating at the ruffled border, we have combined biochemical methods with high-resolution tandem mass spectrometry (LC-MS/MS) to unbiasedly survey the osteoclast lysosomal membrane proteome using isolated secretory lysosomes as a surrogate. Using this approach, we unambiguously identified 2351 unique proteins, of which 421 were enriched on secretory lysosomes, including 102 unique IDs functionally assigned as membrane transporters. These transporters include all subunits of the V-ATPase proton pumps, chloride ion channels, secondary active transporters of the Solute carrier (Slc) protein superfamily, and others whose localisations are predicted to reside on lysosomes, but their functions remain unknown. By combining a suite of biochemical, cell biology and genetic studies, we demonstrated the robustness and utility of our proteomic screen using the Slc37a2 transporter as a prototype. This approach has allowed us, for the first time, to unmask the entire osteoclast lysosomal transporter cache (termed the ‘Transportome’), that will serve as a powerful resource for the future interrogation of ‘orphan’ lysosomal transporters, whose physiological functions in osteoclasts and bone homeostasis have yet to be ascribed.