Demonstration of Lysosomal Localization for the Mammalian Ependymin-related Protein Using Classical Approaches Combined with a Novel Density Shift Method

Abstract

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.Recent genomic and proteomic studies have revealed the presence of numerous previously uncharacterized proteins encoded by the mammalian genome. Determining the biological role of such proteins poses a fundamental challenge. Although sequence and structural similarities to proteins of known function may provide useful information, knowledge of the site of function is essential in understanding uncharacterized proteins. In addition to providing insight into protein function, mapping of proteins to cellular organelles or structures involved in specific biological processes may provide valuable clues to how the organelles carry out these processes.One cellular compartment that contains protein constituents of characteristic function is the lysosome, an acidic, membrane-delimited organelle present in all eukaryotic cells (1). The lysosome contains over 60 soluble proteins, most of which are hydrolases that function in concert to degrade macromolecules introduced via the endocytic and autophagic pathways into simple constituents that can be reutilized by the cell. A number of recent proteomic studies investigating soluble luminal lysosomal proteins have relied upon the fact that these proteins contain an unusual carbohydrate modification that allows for their specific affinity purification. Most luminal lysosomal proteins are t...

Loss of Niemann-Pick C1 or C2 Protein Results in Similar Biochemical Changes Suggesting That These Proteins Function in a Common Lysosomal Pathway

“…In the brain, the absence of either NPC1 or NPC2 had no significant effect on the relative distribution of the lysosomal markers (Fig. 3), as noted previously for NPC2 [28]. This differs from other reports [32] but may reflect the fact that we analyze the complete brain, rather than discrete regions.…”

“…If both NPC1 and NPC2 proteins function in a common pathway, deficiencies in either should produce similar effects on the biophysical properties of lysosomes. Previously, we conducted subcellular fractionation of brains from symptomatic Npc2 mutants and found significant decrease in the buoyant density of the lysosome but not in other cellular organelles [28]. Others have found a decrease in the buoyant density of lysosomes in the liver, spleen and lung from symptomatic Npc1 -mutant animals [29], [30].…”

“…Densities of all the fractions were measured by refractometry and samples were stored at −80°C prior to use. Enzyme and protein assays were performed as described previously [28], [44], [47].…”

Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes

“…Epdr1 probably encodes a lysosomal protein [57] homologous to human UCC1/MERP1 [58]. In both models, Epdr1 transcript levels did not significantly change after T. gondii infection (data not shown).…”

Residual levels of tripeptidyl-peptidase I activity dramatically ameliorate disease in late-infantile neuronal ceroid lipofuscinosis

“…Brain and liver homogenates (H fraction) were prepared using a Potter homogenizer as described [14] in 0.25M sucrose. Nuclei and heavy mitochondrial components were pelleted by centrifugation for 3 minutes at 12,500 rpm in a 50 Ti rotor (Beckman Coulter, Fullerton, CA).…”