Organelle Number is Determined Solely by Coat-SNARE Interactions in a Computational Model of the Eukaryotic Endomembrane System

Abstract

The endoplasmic reticulum is permeable to a variety of small molecules, ranging from calcium to sugars and small peptides. We previously reported that constitutively-open, Sec61 translocons provide a non-selective pathway for small molecules to cross the ER membrane (Pflugers Arch., 457:917). The goal of the present study was to determine the size of the pathway through which small molecules passively cross the ER membrane by comparing the relative permeabilities of a series of neutral sugars, including ribose, galactose, sucrose, and raffinose. Rough ER microsomes from pig pancreas were immobilized on a coverslip, and a low-power, total internal reflection microscope was used to detect changes in light scattering produced when the flowing bath solution was changed from 140 mM K-Acetate/2.5 mM MgCl2/10 mM HEPES to the same solution supplemented with 100 mM of one of the sugars. All of the sugars produced an increase in scattering that recovered exponentially, with the rate of the recovery inversely proportional to the Stokes radius of the sugar. The time constants for recovery were fitted using a Bungay-Brenner function, which models a pore as a simple cylinder and permeation as diffusion without specific interaction of a permeant molecule with the wall of a pore. The relative permeabilities of the four sugars were fitted very well by the Bungay-Brenner function, yielding an average pore diameter of 15 angstroms. Thus, there appears to be a pathway for these sugars to cross the ER membrane that is constitutively active and can be modeled as a 15 angstrom pore. Furthermore, the release of nascent chains by puromycin increased the rate of recovery, whereas treatment with EDTA, which strips ribosomes from the ER membrane, decreased the rate of recovery. Both effects are consistent with the Sec61 translocon contributing to this pathway.