Intracellular trafficking of the JNCL protein CLN3
| Autor: | Beverly L. Davidson, Todd A. Derksen, Ronald E. Haskell |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Endocrinology Diabetes and Metabolism Recombinant Fusion Proteins Green Fluorescent Proteins Immunoblotting Golgi Apparatus Biology Endoplasmic Reticulum Transfection Biochemistry Coatomer Protein Models Biological Green fluorescent protein chemistry.chemical_compound symbols.namesake Endocrinology Neuronal Ceroid-Lipofuscinoses Cyclins Genetics Humans Molecular Biology Peptide sequence Cells Cultured Brefeldin A Membrane Glycoproteins Rhodamines Endoplasmic reticulum HEK 293 cells Golgi apparatus Fusion protein Molecular biology Immunohistochemistry Cell biology Luminescent Proteins CLN3 chemistry symbols Microtubule-Associated Proteins Molecular Chaperones |
| Zdroj: | Molecular genetics and metabolism. 66(4) |
| ISSN: | 1096-7192 |
| Popis: | Juvenile neuronal ceroid lipofuscinosis is a lysosomal storage disease that causes visual impairment, progressive mental deterioration, and eventually death. A predominant 1.02-kb deletion as well as other mutations have been described in the CLN3 gene. Lacking significant identity with proteins of known function and no overt targeting signals within the primary amino acid sequence, accurate predictions of the intracellular location and function could not be made. Further, recent conflicting reports identified CLN3 as either a lysosomal or a mitochondrial protein. Transfection experiments using native and epitope-tagged fusion proteins were evaluated to help delineate CLN3 localization. We confirmed by immunohistochemistry and brefeldin A treatment that NH2-terminal green fluorescence protein (GFP)-CLN3 fusion proteins were retained in the Golgi apparatus, with no colocalization with mitochondrial markers. Anti-CLN3 antibodies directed against amino acids 67-90 of CLN3 were generated and shown to be specific for a 50-kDa protein in HEK 293 cells and GFP-CLN3 in transfected cells. However, cells transfected with nontagged CLN3 or carboxyl-terminal-tagged CLN3 were not immunoreactive with anti-CLN3 antibodies, suggesting that normally, the amino terminus interacts with other molecules. Thus, tags on the NH2-terminus probably inhibited these interactions and movement of CLN3 from the Golgi to more distal compartments. Also, CLN3 tagged at the COOH-terminus with either GFP or FLAG epitopes were retained in the ER, indicating a role for the COOH-terminus in trafficking. Taken together, these data confirm that CLN3 traffics through the ER and Golgi. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect