Mannose 6-phosphate (Man6P) residues represent a recognition signal required for 
efficient receptor-dependent transport of soluble lysosomal proteins to 
lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice 
deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both 
phosphatases (Acp2/Acp5(-/-)) to examine their role in dephosphorylation of 
Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of 
tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting 
in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The 
most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P 
affinity chromatography and mass spectrometry. Depending on the presence of Acp2 
or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 
ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively 
charged lysosomal membranes at acidic pH. Correspondingly, unesterified 
cholesterol was found to accumulate in lysosomes of cultured hepatocytes of 
Acp2/Acp5(-/-) mice. The data demonstrate that dephosphorylation of 
Man6P-containing lysosomal proteins requires the concerted action of Acp2 and 
Acp5 and is needed for hydrolysis and removal of degradation products.