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Mehdi Najafi, Nycole A. Maza, Peter D. Calvert; Interplay Between Steric Volume Exclusion and Posttranslational Lipidation in Setting Protein Concentrations in Photoreceptor Sensory Cilia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4130.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that the proportions of soluble molecules found in the ciliary rod outer segment (OS), relative to the inner segment (IS), were steeply dependent on molecular size and the geometry of cytoplasmic spaces due to steric volume exclusion (SVE)1. Since it is widely believed that the majority of (OS) proteins involved in light signaling are targeted to and confined there by addition of lipid moieties, we systematically evaluated how posttranslational modifications and SVE act together to set the distributions of proteins in rods.
Concatamers of EGFP (1x-, 2x- and 3xEGFP), without and with lipid modifications, including single myristoyl (Myr), farnesyl (Far) or geranylgeranyl (GerGer), double geranylgeranyl (EGFP-(GerGer)2) or myristoyl and farnesyl (Myr-EGFP-Far), were expressed in Xenopus laevis rods. Protein distributions in live retinal slices were determined from 3D confocal scans. To study protein mobility, PAGFP variants of the probes were locally multiphoton-photoactivated and their movements subsequently monitored by confocal imaging.
Single-lipidated EGFPs are present in the OS at as much as 40% greater abundance compared to 1xEGFP. The distribution of Myr-EGFP-Far was similar to the distribution of single-lipidated EGFPs whereas EGFP-(GerGer)2 was predominantly localized to the OS. Mobility studies show that single-lipidated PAGFPs and Myr-PAGFP-Far were able to dissociate from membranes and travel between compartments whereas PAGFP-(GerGer)2 was tightly membrane-bound, never dissociating from membranes over the course of hours.
Addition of lipid moieties to GFPs variably overcame SVE, resulting in increased OS abundance. Tight membrane binding via addition of (GerGer)2 can confine proteins to the OS, but addition of single lipid moieties that are common to any phototransduction proteins, including transducin, rhodopsin kinase and PDE6, are not sufficient to do so. Finally, we propose a novel mechanism for light-dependent translocation of transducin to the IS whereby transducin subunits associate with partners that act to increase their solubility as well as their effective size, thus driving transport to the IS via SVE.1Najafi et al., Proc. Natl. Acad. Sci.,in press.
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