Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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).
Keywords: anterior segment • protein structure/function • proteins encoded by disease genes