Scrg1, a novel protein of the CNS is targeted to the large dense-core vesicles in neuronal cells.
Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine-rich protein 98 amino acids in length, with a N-terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense-core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense-core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense-core vesicles, as indicated by immuno-electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense-core vesicle-associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.
Dandoy-Dron
Françoise
F
Laboratoire d'Oncologie Virale, CNRS UPR 9045, 7 rue G. Môquet BP-8, 94801 Villejuif Cedex, France.
Griffond
Bernadette
B
Mishal
Zohar
Z
Tovey
Michael G
MG
Dron
Michel
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002490:Central Nervous System / Q000737:chemistry; D002864:Chromogranins; D018076:DNA, Complementary; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D009474:Neurons / Q000737:chemistry*; D011506:Proteins / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D013572:Synaptic Vesicles / Q000737:chemistry*; D050857:Synaptotagmins
D000906:Antibodies; D002135:Calcium-Binding Proteins; D002864:Chromogranins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; C111496:Scrg1 protein, mouse; C494296:Syt5 protein, mouse; D050857:Synaptotagmins