PMID- 16954209
OWN - NLM
STAT- MEDLINE
DCOM- 20070118
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 281
IP  - 46
DP  - 2006 Nov 17
TI  - Demonstration of lysosomal localization for the mammalian ependymin-related 
      protein using classical approaches combined with a novel density shift method.
PG  - 35436-45
AB  - 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.
FAU - Della Valle, Maria Cecilia
AU  - Della Valle MC
AD  - 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.
FAU - Sleat, David E
AU  - Sleat DE
FAU - Sohar, Istvan
AU  - Sohar I
FAU - Wen, Ting
AU  - Wen T
FAU - Pintar, John E
AU  - Pintar JE
FAU - Jadot, Michel
AU  - Jadot M
FAU - Lobel, Peter
AU  - Lobel P
LA  - eng
GR  - DK054317/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20060905
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Mannosephosphates)
RN  - 0 (Nerve Tissue Proteins)
RN  - 0 (Npc2 protein, mouse)
RN  - 0 (Vesicular Transport Proteins)
RN  - 0 (ependymins)
RN  - 3672-15-9 (mannose-6-phosphate)
SB  - IM
MH  - Animals
MH  - Brain/metabolism
MH  - Gene Deletion
MH  - Lipidoses/genetics/metabolism
MH  - Lysosomes/*metabolism
MH  - Mannosephosphates/metabolism
MH  - Mice
MH  - Nerve Tissue Proteins/*metabolism
MH  - Phosphorylation
MH  - Pichia
MH  - Rats
MH  - Vesicular Transport Proteins/metabolism
EDAT- 2006/09/07 09:00
MHDA- 2007/01/19 09:00
CRDT- 2006/09/07 09:00
PHST- 2006/09/07 09:00 [pubmed]
PHST- 2007/01/19 09:00 [medline]
PHST- 2006/09/07 09:00 [entrez]
AID - S0021-9258(19)35021-5 [pii]
AID - 10.1074/jbc.M606208200 [doi]
PST - ppublish
SO  - J Biol Chem. 2006 Nov 17;281(46):35436-45. doi: 10.1074/jbc.M606208200. Epub 2006 
      Sep 5.