Demonstration of lysosomal localization for the mammalian ependymin-related protein using classical approaches combined with a novel density shift method.
Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.
Della Valle
Maria Cecilia
MC
Center for Advanced Biotechnology and Medicine, Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Sleat
David E
DE
Sohar
Istvan
I
Wen
Ting
T
Pintar
John E
JE
Jadot
Michel
M
Lobel
Peter
P
D000818:Animals; D001921:Brain / Q000378:metabolism; D017353:Gene Deletion; D008064:Lipidoses / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010843:Pichia; D051381:Rats; D033921:Vesicular Transport Proteins / Q000378:metabolism
D008360:Mannosephosphates; D009419:Nerve Tissue Proteins; C497045:Npc2 protein, mouse; D033921:Vesicular Transport Proteins; C031632:ependymins; C027693:mannose-6-phosphate