| Abstract No.: | A-C1110 |
| Country: | Canada |
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| Title: | SCYL1, MUTATED IN A RECESSIVE FORM OF SPINOCEREBELLAR ATAXIA, REGULATES COPI-MEDIATED RETROGRADE TRAFFIC |
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| Authors/Affiliations: | 1 Jonathon Burman*; 1 Lyne Bourbonniere; 1 Thomas Stroh; 2 John Presley; 1 Peter McPherson;
1 Montreal Neurological Institute, QC, Canad; 2 McGill University, Montreal, QC, Canada
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| Content: | Objectives: Scy1-like 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases was recently identified as the gene product altered in muscle deficient mice (mdf), which suffer from motor neuron degeneration, Purkinje cell loss and cerebellar atrophy. To determine the function of Scyl1 we have undertaken a biochemical and cell biological characterization of the protein.
Materials and Methods: We have used standard cell biological and biochemical assays including affinity selection experiments coupled to mass spectrometry for the discovery of novel protein-protein interactions, indirect immunofluorescence microscopy in cell lines and primary hippocampal cultures, peptide competition assays, immunoprecipitations and analysis of cells following treatment with nocodazole and brefeldin A. We also examined anterograde and retrograde trafficking through the secretory pathway using exogenously expressed vesicular stomatitis virus glycoprotein (VSVG), either alone or coupled to the KDEL receptor.
Results: Affinity selection experiments in conjunction with mass spectrometry identified bands co-purifying with Scyl1 as components of the coatomer I (COPI) complex. The interaction was confirmed in pull-down assays and Scyl1 co-immunoprecipitates with COPI coats from tissue lysates. Interestingly, and unique for a non-transmembrane domain protein, Scyl1 binds COPI coats using a C-terminal RKLD-COO- sequence, similar to the KKXX-COO- COPI-binding motif found in transmembrane ER proteins. Scyl1 co-localizes with COPI coat proteins and is localized, in an Arf1-independent manner, to the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, sites of COPI-mediated membrane budding. The localization and binding properties of Scyl1 strongly suggest a function in COPI transport. Importantly, depletion of Scyl1 using small interfering RNAs, mimicking the nonsense mediated mRNA degradation observed in mdf mice, results in a defect in retrograde trafficking of the KDEL receptor from the cis-Golgi to the ER. Together, these studies place Scyl1 at an interface between COPI-coated vesicles and the membranes of the early secretory pathway, where it functions in COPI-mediated retrograde trafficking.
Conclusion: This work adds significant information to the function of the Scyl1 protein, mutated in a mouse model of spinocerebellar neurodegeneration. We find that Scyl1 functions in COPI-mediated retrograde trafficking from the cis-Golgi and ERGIC to the ER. Our data support the retrograde directionality of at least a subset of COPI-coated structures, and present Scyl1 as the first cytosolic protein to utilize a dibasic motif to bind COPI and function as an accessory protein in this trafficking pathway. Our results also provide the first indication that mutations in the COPI pathway contribute to a neurodegenerative disorder.
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