Lysine is a common determinant for mannose phosphorylation of lysosomal proteins.
The phosphorylation of lysosomal enzymes on high mannose residues is the first step in the targeting of these enzymes to lysosomes in a wide range of mammalian cells. Phosphorylated lysosomal enzymes bind to mannose 6-phosphate receptors, which divert them from the secretory pathway and direct them toward the lysosome. We have been investigating the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase by using the precursor form of the lysosomal cysteine protease, cathepsin L, as a model lysosomal enzyme in an in vitro assay for mannose phosphorylation. Cathepsin L was found to be phosphorylated in vitro with the same efficiency as other lysosomal enzymes and to contain a conformationally sensitive protein signal that is recognized by phosphotransferase. Biochemical modification of lysine residues on cathepsin L with sulfo-N-hydroxysuccinimide acetate prevented the enzyme from being phosphorylated, indicating that lysine is an important component of the signal. The modification itself did not cause any major conformational changes in cathepsin L. When the same modification was performed on a number of other lysosomal enzymes, phosphorylation was also inhibited. Thus, we conclude that lysine residues are important features of lysosomal enzyme phosphotransferase recognition sites in general, and we discuss the implications of this finding in the ongoing efforts to define the phosphotransferase recognition site.
Cuozzo
J W
JW
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Sahagian
G G
GG
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000378:metabolism; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D008247:Lysosomes / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
D004792:Enzyme Precursors; D006023:Glycoproteins; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D008239:Lysine; D008358:Mannose