Saposins Aa nd Ca re sphingolipid activator proteins requiredf or thel ysosomal breakdowno f galactosylceramide and glucosylceramide,r espectively.T he saposins interact with lipids, leading to an enhanced accessibility of thel ipid headgroups to their cognate hydrolases. We have determinedt he crystal structures of humans aposinsAandCt o2 .0 A˚and 2.4Å ,r espectively,a nd both reveal the compact, monomeric saposin fold. We confirmed that these twoproteins were monomeric in solution at pH 7.0b yanalytical centrifugation. However, at pH 4.8,inthe presence of thedetergent C 8 E 5 ,saposin Aa ssembled intod imers, while saposin Cf ormed trimers. Saposin Bw as dimeric under allc onditions tested.T he self-associationo ft he saposins is likelyt ob er elevantt oh ow theses mall proteins interact with lipids, membranes, andh ydrolase enzymes.Keywords: saposins;X -ray crystallography; analyticalu ltracentrifugation; protein-detergent interactions Saposins A, B, C, and Da re small, nonenzymatic proteins required for the breakdown of glycosphingolipids within the lysosome (Kolter and Sandhoff2005). They are derived from the proteolytic processing of the precursor protein prosaposin, producing the four individual saposins. The four saposin domains contained within prosaposin most likely arose from two tandem duplications of an ancestral gene into one single copy gene (Hazkani-Covoetal. 2002). Each saposin ''activates''the breakdown of particular lipid substrates by facilitating the access of the lipid headgroups to the active sites of cognate hydrolases. It is generally believed that in the absence of sphingolipid activator proteins, the oligosaccharide chains of the membranebound lipids do not extend far enough into the lysosomal lumen to be accessible to the active sites of the hydrolases. Mechanistically,t he saposins appear to activate lipid hydrolysis by solubilizing the lipid substrates or possibly by destabilizing the membrane structure (Vaccaro et al. 1993(Vaccaro et al. , 1995(Vaccaro et al. , 1997Wilkening et al. 1998;Salvioli et al. 2000). Article publishedonline aheado fp rint.A rticle and publicationd ate area th ttp://www.proteinscience.org/cgi