Mechanisms of protein transport between subcompartments of the cone photoreceptor outer segment membrane system

(≤300 words, current = 296): Retinal cone photoreceptor outer segments (COS) are modified cilia where light is transduced into electrical signals. While extensive ultrastructural studies have established that the COS membrane system consists of several hundred parallel lamellae interconnected by the ciliary plasma membrane (PM) through narrow membrane bridges known as saddle points (SP), little is known about how components of the phototransduction cascade are delivered to COS, how they are loaded into flat lamellae surfaces (LS) and how they maintain uniform distribution among lamellae, especially in light of the steeply tapered lamellar geometry found in some species and high membrane turnover rate. Here, we quantify interlamellar membrane protein flux in Xenopus COS using a neutral membrane probe - the transmembrane domain of syntaxin 3 fused with EGFP (EGFP-Stx3TM), and develop 3D and 1D computational models of protein transport within the COS membrane system. The models describe diffusional transport of the probes along the folded membranes accounting for effects of molecular crowding. Axial equilibration of EGFP-Stx3TM in FRAP experiments is quantitatively described by the model based on diffusional transport, alone. The diffusion coefficient on the PM obtained from the model fitted to experimental data is approximately equal to that on the LS. Sensitivity analysis of the model shows that, surprisingly, SP contribute only ∼15% control of axial transport while the chief control, ∼80%, is located at the PM, and this control distribution is robust to changes in conditions. Thus, COS plasma membrane acts as a shunt conducting free transmembrane proteins between lamellae by Brownian diffusion, even in the presence of crowding effects and complex membrane geometry, while lamellae are capacitive sources/sinks. Axial Stx3TM flux can exceed 100 molecules/s suggesting that if opsins diffuse freely, their flux may exceed 104 molecules/s, supporting rapid equilibration and uniform distribution throughout the contiguous COS membrane system.