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William M. Dismuke, Brian S. McKay, W Daniel Stamer; Myocilin, Q-snare Homology And Membrane-associated Binding Partners. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2068.
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Mutations in the widely expressed protein myocilin result in a specific phenotype, ocular hypertension, glaucoma and blindness, yet the function of myocilin remains controversial and elusive. Our preliminary data suggest that membrane-associated myocilin is present in a large protein complex. Here we test the hypothesis that myocilin is a component of a SNARE complex.
We first used sequence analysis to examine structural homology of myocilin to known SNARE components. Second we used five complementary biochemical methods to examine the hydrodynamic properties of the myocilin complex; rotary shadowing, velocity gradient sedimentation, gel exclusion chromatography, immunoprecipitation and western blotting.
Our sequence analysis of the coiled-coil domain in a number of mammals revealed a segment with a hydrophobic stripe that exhibits homology to highly conserved regions of known Q-SNAREs. We found myocilin to exist in two cellular pools when separated by PAGE; as a 55/57kDa monomer in the soluble fraction and as a large, SDS-resistant protein complex in the membrane fraction. From the membrane fraction, velocity gradient sedimentation showed myocilin separating as small dimer (6.4s) and a large complex (17.3s). Myocilin was detected in a complex separating at 405-440 kDa by gel exclusion chromatography. Immunoprecipitation showed an association of myocilin with another coiled-coil containing SNARE protein, VAMP 1.
Our data demonstrates that membrane-associated myocilin is present in a large, globular, detergent-resistant protein complex similar to other SNARE complexes (Söllner T, et al. Nature, 1993). The Q-SNARE homology region identified within the coiled-coil may facilitate the interaction of myocilin with its binding partners (Chapman ER, et al. JBC 1994).
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