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• W2017988657 abstract "The pore of the translocon complex in the endoplasmic reticulum (ER) is large enough to be permeated by small molecules, but it is generally believed that permeation is prevented by a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl α-d-glucopyranoside (4MαG), a small, neutral dye molecule, cannot permeate an empty translocon pore by measuring its activation by an ER resident α-glucosidase, which is dependent on entry into the ER. The basal entry of dye into the ER of broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores. The basal and puromycin-dependent entries of 4MαG were mediated by a common, salt-sensitive pathway that was partially blocked by spermine. A similar activation of 4MαG was observed in nystatin-perforated cells, indicating that the entry of 4MαG into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject the hypothesis and conclude that a small, neutral molecule can permeate the empty pore of a translocon complex, and we propose that translationally inactive, ribosome-bound translocons could provide a pathway for small molecules to cross the ER membrane. The pore of the translocon complex in the endoplasmic reticulum (ER) is large enough to be permeated by small molecules, but it is generally believed that permeation is prevented by a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl α-d-glucopyranoside (4MαG), a small, neutral dye molecule, cannot permeate an empty translocon pore by measuring its activation by an ER resident α-glucosidase, which is dependent on entry into the ER. The basal entry of dye into the ER of broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores. The basal and puromycin-dependent entries of 4MαG were mediated by a common, salt-sensitive pathway that was partially blocked by spermine. A similar activation of 4MαG was observed in nystatin-perforated cells, indicating that the entry of 4MαG into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject the hypothesis and conclude that a small, neutral molecule can permeate the empty pore of a translocon complex, and we propose that translationally inactive, ribosome-bound translocons could provide a pathway for small molecules to cross the ER membrane. endoplasmic reticulum immunoglobulin-binding protein 4-methylumbelliferyl α-d-glucopyranoside 4-methylumbelliferyl β-d-glucopyranoside Chinese hamster ovary-S cells initial slope basal, puromycin-independent slope pur, slope in the presence of puromycin pur, increase ofS0,pur above S0,basal maximum number of empty pores in the presence of puromycin background activation The endoplasmic reticulum (ER)1 is the site of essential synthetic and signaling processes, such as the synthesis of secreted and membrane proteins and the production of calcium signals. These processes require the transport of a broad spectrum of molecules across the ER membrane while maintaining a selectivity during transport which prevents the loss of essential gradients. The ER membrane is rich in pathways for the active and passive transport of molecules ranging in size from ions and small polar molecules to proteins. The pore of the translocon (sec61 complex) is the largest pore in the ER membrane, with an estimated diameter of 40–60 Å in the ribosome-bound state and a smaller diameter of 9–15 Å in the ribosome-free state (1Hamman B.D. Chen J.C. Johnson E.E. Johnson A.E. Cell. 1997; 89: 535-544Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 2Hamman B.D. Hendershot L.M. Johnson A.E. Cell. 1998; 92: 747-758Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). When bound by a ribosome, the pore of the translocon is aligned with the peptide exit tunnel in the large subunit of the ribosome (3Beckmann R. Bubeck D. Grassucci R. Penczek P. Verschoor A. Blobel G. Frank J. Science. 1997; 278: 2123-2126Crossref PubMed Scopus (292) Google Scholar), and the average diameter of this tunnel is about 20 Å (4Morgan D.G. Menetret J.F. Radermacher M. Neuhof A. Akey I.V. Rapoport T.A. Akey C.W. J. Mol. Biol. 2000; 301: 301-321Crossref PubMed Scopus (53) Google Scholar). Together, the polypeptide exit tunnel of the ribosome and the pore of the translocon provide a linked pore of sufficiently large dimensions to permit the translocation of polypeptides across the ER membrane. The role of the translocon pore as the pathway used for the cotranslational insertion of nascent proteins into the ER is well established, and recent evidence supports an even broader role for the translocon pore in protein transport because the pore also provides a pathway for the retrograde export of proteins from the lumen of the ER to the cytosol (5Gillece P. Pilon M. Romisch K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4609-4614Crossref PubMed Scopus (36) Google Scholar, 6Romisch K. J. Cell Sci. 1999; 112: 4185-4191Crossref PubMed Google Scholar). A model for ribosome-translocon interactions in which the large subunit of the ribosome remains bound to the translocon pore after translation is terminated was proposed recently (7Potter M. Seiser R.M. Nicchitta C.V. Trends Cell Biol. 2001; 11: 112-115Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). An intriguing implication of this model is that the ER membrane might contain a large number of ribosome-bound translocon complexes with the pore unoccupied by polypeptides. The very large diameter of the pore of the ribosome-translocon complex raises the possibility that many small molecules could permeate this pathway when it is empty, i.e. unoccupied by polypeptides. According to the prevailing model, a large barrier to permeation of the translocon pore is produced by the binding of BiP, a prominent ER chaperone protein, to the luminal end of the pore (2Hamman B.D. Hendershot L.M. Johnson A.E. Cell. 1998; 92: 747-758Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 8Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (501) Google Scholar). This gate can be opened only by nascent polypeptides that have grown to a length greater than ≈70 amino acids. However, this model was developed using charged molecules to probe the permeability of the translocon pore, and the question remains whether the binding of BiP to the pore actually produces a tight mechanical seal that blocks the passage of all molecules, or, alternatively, does the binding of BiP produce a looser seal that functions as a selectivity filter, allowing some molecules to pass through? In particular, is there a barrier to permeation of the translocon pore by neutral, polar molecules? The answer to this question is important because permeation of the translocon pore by small neutral molecules could play an essential role in bidirectional signaling or the transport of substrates between the lumen of the ER and the cytosol. We have tested the hypothesis that the translocon pore maintains a high barrier to permeation by neutral, as well as charged molecules by measuring the entry into the ER of a small polar molecule, 4-methylumbelliferyl α-d-glucopyranoside (4MαG). The rationale for this assay was that entry of 4MαG into the ER can be detected if it is cleaved by α-glucosidase II, a resident ER glucosidase (9Burns D.M. Touster O. J. Biol. Chem. 1982; 257: 9990-10000PubMed Google Scholar, 10Brada D. Dubach U.C. Eur. J. Biochem. 1984; 141: 149-156Crossref PubMed Scopus (72) Google Scholar, 11Hino Y. Rothman J.E. Biochemistry. 1985; 24: 800-805Crossref PubMed Scopus (32) Google Scholar, 12Strous G.J. Van Kerkhof P. Brok R. Roth J. Brada D. J. Biol. Chem. 1987; 262: 3620-3625Abstract Full Text PDF PubMed Google Scholar, 13Saxena S. Shailubhai K. Dong-Yu B. Vijay I.K. Biochem. J. 1987; 247: 563-570Crossref PubMed Scopus (27) Google Scholar, 14Lucocq J.M. Brada D. Roth J. J. Cell Biol. 1986; 102: 2137-2146Crossref PubMed Scopus (74) Google Scholar), with the release of a fluorescent product. The rate of accumulation of the fluorescent product should then provide a measure of the rate of entry of 4MαG into the ER. α-Glucosidase II has been purified from the ER (7Potter M. Seiser R.M. Nicchitta C.V. Trends Cell Biol. 2001; 11: 112-115Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 9Burns D.M. Touster O. J. Biol. Chem. 1982; 257: 9990-10000PubMed Google Scholar, 10Brada D. Dubach U.C. Eur. J. Biochem. 1984; 141: 149-156Crossref PubMed Scopus (72) Google Scholar, 12Strous G.J. Van Kerkhof P. Brok R. Roth J. Brada D. J. Biol. Chem. 1987; 262: 3620-3625Abstract Full Text PDF PubMed Google Scholar, 13Saxena S. Shailubhai K. Dong-Yu B. Vijay I.K. Biochem. J. 1987; 247: 563-570Crossref PubMed Scopus (27) Google Scholar, 15Trombetta E.S. Simons J.F. Helenius A. J. Biol. Chem. 1996; 271: 27509-27516Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). It is a soluble, heterodimeric enzyme that contains an HEDL ER retention signal (15Trombetta E.S. Simons J.F. Helenius A. J. Biol. Chem. 1996; 271: 27509-27516Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), and immunohistochemical staining has demonstrated that it is specifically expressed in the lumen of the ER and some transitional elements of the ER (14Lucocq J.M. Brada D. Roth J. J. Cell Biol. 1986; 102: 2137-2146Crossref PubMed Scopus (74) Google Scholar). α-Glucosidase II specifically cleaves α1,3-glycosidic linkages, and its activity is optimal at pH 6.5–7.0 (9Burns D.M. Touster O. J. Biol. Chem. 1982; 257: 9990-10000PubMed Google Scholar, 10Brada D. Dubach U.C. Eur. J. Biochem. 1984; 141: 149-156Crossref PubMed Scopus (72) Google Scholar, 13Saxena S. Shailubhai K. Dong-Yu B. Vijay I.K. Biochem. J. 1987; 247: 563-570Crossref PubMed Scopus (27) Google Scholar, 15Trombetta E.S. Simons J.F. Helenius A. J. Biol. Chem. 1996; 271: 27509-27516Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 16Kaushal G.P. Pastuszak I. Hatanaka K. Elbein A.D. J. Biol. Chem. 1990; 265: 16271-16279Abstract Full Text PDF PubMed Google Scholar), which clearly distinguishes it from the acidic α-glucosidase found in the lysosomes of some cells (17Van Hove J.L. Yang H.W. Wu J.Y. Brady R.O. Chen Y.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 65-70Crossref PubMed Scopus (105) Google Scholar). The activity of α-glucosidase II can also be distinguished from α-glucosidase I, another neutral ER glucosidase (18Shailubhai K. Pukazhenthi B.S. Saxena E.S. Varma G.M. Vijay I.K. J. Biol. Chem. 1991; 266: 16587-16593Abstract Full Text PDF PubMed Google Scholar, 19Shailubhai K. Pratta M.A. Vijay I.K. Biochem. J. 1987; 247: 555-562Crossref PubMed Scopus (43) Google Scholar), by the fact that 4MαG is not hydrolyzed by α-glucosidase I (19Shailubhai K. Pratta M.A. Vijay I.K. Biochem. J. 1987; 247: 555-562Crossref PubMed Scopus (43) Google Scholar). We report that the empty translocon pore is highly permeable to 4MαG, and we propose that translationally inactive ribosome-translocon complexes might provide a pathway for the movement of small, neutral molecules across the ER membrane. 4MαG and puromycin HCl were from Calbiochem. All other reagents were from Sigma. CHO-S cells (Life Technologies, Inc.) were grown to a density of 0.5–1.0 × 106 cells/ml in stirrer flasks in serum-free medium (Life Technologies, Inc.) at 37 °C in 5% CO2. For a 32-well assay (eight conditions in quadruplicate), 20 ml of cells was pelleted and resuspended in 20 ml of KG buffer (140 mm potassium glutamate, 2.5 mm MgCl2, 10 mmHEPES, pH 7.25). The resuspended cells were pressurized for 2 min at 80 p.s.i. of N2 in a Parr nitrogen cavitation homogenizer and gently broken open by release through the needle valve. Under these conditions, 53% of the cells were permeabilized, as measured by propidium iodide staining. Gentle opening of the plasma membrane was essential. Increasing the N2 cavitation pressure produced a disproportionate increase in the base-line activation of 4MαG, a likely result of increased breakage of the ER with the more vigorous cavitation. Permeabilization with 100 μm digitonin, which was 100% effective, increased the variability of our measurements, perhaps as a result of destabilization of the ER membrane by the detergent. We also observed that any attempt to pellet the broken cells produced a greatly increased background fluorescence. Assays were performed using Nunc 48-well plates read in a CytoFluor 4000 (PE Biosystems) plate reader. 0.5 ml of a suspension of broken cells was loaded per well. A stock solution of 20 mm 4MαG was prepared in methanol and diluted into KG buffer at a final concentration of 20 μm, unless otherwise noted. The center wavelength and bandwidth of the excitation and emission filters were 360/40 and 460/40, respectively. All measurements were made at 35–37 °C. The plate with solutions was prewarmed to 37 °C for 15 min, and the dye was added immediately before transferring the plate to the reader. The fluorescence was measured for 30 min at 2-min intervals, with 10 s of mixing before each measurement. Puromycin HCl was prepared as a 10 mmstock solution in water and used at a final concentration of 100 μm. The substrate 4MαG is nonfluorescent until the glycosidic linkage between the glucose and coumarin dye moieties is cleaved by α-glucosidase, releasing the free, fluorescent dye. The activation of 4MαG is irreversible, and the slope of the fluorescenceversus time curve, S(t), is proportional to the rate of activation of the dye at timet. Under most conditions,S(t) was a constant, but under some conditions there was a gradual increase or decrease inS(t) which followed a simple exponential time course. The linear and exponential contributions toS(t) were well fitted by Equation1 S(t)=S0*ek*tEquation 1 where S0 is the initial slope (ΔF min−1)and k is an exponential rate constant (min−1). Best fit estimates of S0 and k for each well were obtained using the Solver nonlinear curve-fitting routine in Excel (Microsoft). Parameters for Michaelis-Menten and Hill functions were estimated using the Levenberg-Marquardt nonlinear curve-fitting routine in Origin, version 6.0 (Microcal). Statistical analysis was performed using JMPin (SAS Institute). Averages are plotted ± S.E. of the mean. We first examined the activation of 4MαG in a broken cell preparation nonspecifically permeabilized by the addition of 0.05% sodium deoxycholate. Representative data and fitted curves calculated from Equation 1 are shown in Fig.1 A. The increase in fluorescence over time was fitted well by a combination of linear and exponential components, with the linear component dominating at concentrations of 4MαG above 10 μm. At lower concentrations of 4MαG, an exponential decay of the initial slope,S0, was apparent, probably as a result of depletion of the 4MαG substrate with time. There was no activation of dye when 4MαG was added to KG buffer in the absence of cells (data not shown). The dependence of S0 on the concentration of 4MαG is shown in Fig. 1 B. At 4MαG concentrations below 100 μm, S0values were fitted by a single Michaelis-Menten function with aKm of ≈5 μm, which was close to theKm of α-glucosidase II for 4MαG reported in bovine mammary gland (10Brada D. Dubach U.C. Eur. J. Biochem. 1984; 141: 149-156Crossref PubMed Scopus (72) Google Scholar). Although the α-glucosidase activity at low substrate concentrations could be characterized by a single kinetically defined process, a second, highly variable component became increasingly evident at concentrations of 4MαG ≥ 100 μm (Fig. 1 B). We did not identify the source of this variable component. We chose 20 μm as a standard working concentration for our experiments because it was below the concentration at which the rate of activation became highly variable, yet it was high enough to drive a sufficient steady-state influx into a membrane-bound compartment (described below). We next examined the rate of activation of 4MαG as a function of pH to determine if 4MαG could be activated in CHO-S cells by an acidic α-glucosidase that is present in the lysosomes of some cells. The activation of 4MαG was strongly dependent on pH, and it was clearly attributable to glucosidase activity that was optimal between pH 6.2 and 7.5. There was no evidence of an acidic α-glucosidase (Fig.2 A), which has a pH optimum of 4.5 (17Van Hove J.L. Yang H.W. Wu J.Y. Brady R.O. Chen Y.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 65-70Crossref PubMed Scopus (105) Google Scholar). We also performed an assay for β-glucosidase, which, if present in CHO-S cells, might nonspecifically activate some 4MαG. Equimolar substitution of 4MαG by 4-methylumbelliferyl β-d-glucopyranoside, the β anomer of 4MαG and substrate for β-glucosidase, yielded no fluorescent product (Fig.2 B). This assay was performed under conditions in which the activity of β-glucosidase added to the preparation was readily detectable (Fig. 2 B). We conclude that neither acidic α-glucosidase nor β-glucosidase is present in CHO-S cells, and 4MαG is activated in CHO-S cells by α-glucosidase II. The addition of 4MαG to the solution bathing intact CHO-S cells resulted in very little activation of the dye (Fig.3 A), indicating that 4MαG does not easily cross intact membranes. To enable access of 4MαG to the ER, we chose low pressure N2 cavitation as a gentle method to break open the plasma membrane. The addition of 4MαG to cavitation-permeabilized cells produced a surprisingly high basal rate of activation, S0,basal, ∼20–45% (median = 28%) of the rate observed in cells permeabilized by 0.05% sodium deoxycholate (Fig. 3 B). This high basal rate was especially remarkable given that cavitation permeabilized only slightly more than 50% of the cells, whereas the detergent permeabilized all of the cells (data not shown). Although a high basal rate of entry of 4MαG into the ER might be inferred from the high rate of activation, an alternate explanation is that 4MαG was activated by α-glucosidase exposed to the buffer by accidental breakage of the ER. We tested this with a protease protection assay using trypsin. When trypsin (0.25% w/v) was added to detergent-permeabilized cells, S(t)decreased exponentially with time (Fig. 3 C), demonstrating that α-glucosidase not protected by intact membranes could be inactivated irreversibly by trypsin. In contrast, the same concentration of trypsin added to cavitation-permeabilized cells in the absence of detergent produced an exponential increase, rather than a decrease, in S(t) (Fig.3 C). The lack of a time-dependent decrease ofS(t) in the absence of detergent demonstrated that the α-glucosidase was protected from proteolysis by trypsin, from which we conclude that the basal activation of 4MαG was produced by α-glucosidase sequestered inside a membrane-bound compartment. Puromycin is an aminoacyl tRNA analog that terminates the elongation of polypeptide chains, releasing nascent polypeptides from ribosomes and clearing the proteins from the pore of the translocon (20Pestka S. Methods Enzymol. 1974; 30: 261-282Crossref PubMed Scopus (55) Google Scholar, 21Skogerson L. Moldave K. Arch. Biochem. Biophys. 1968; 125: 497-505Crossref PubMed Scopus (73) Google Scholar). Puromycin-treated translocons are permeable to ions (22Simon S.M. Blobel G. Cell. 1991; 65: 371-380Abstract Full Text PDF PubMed Scopus (485) Google Scholar), and we tested the prediction that translocon pores cleared of protein by puromycin would provide additional open pores through which 4MαG could enter the ER. The addition of 100 μm puromycin increasedS0 to a value, S0,pur, which was significantly greater than the basal rate (Fig.3 A). The increase in S0 produced by the addition of puromycin, ΔS0,pur =S0,pur − S0,basal, was about 30% greater than S0,basal. We tested puromycin at concentrations between 100 and 500 μm, and we observed maximal dye entry at 100 μm puromycin (data not shown). We conclude from the high specificity of puromycin for terminating translation and the fact that the ER is the only membrane-bound compartment to which ribosomes are attached that the puromycin-dependent activation of 4MαG was produced by the entry of 4MαG into the ER through ribosome-bound translocons. The source of the puromycin-independent, basal activation of 4MαG was less clear. As a first step, we tested by correlation analysis the hypothesis that ΔS0,pur andS0,basal were independent processes. There was a significant correlation (r = 0.49, p < 0.001) between ΔS0,pur andS0,basal (both calculated as a percentage of a detergent control) across a large number of assays (Fig. 3 D,n = 142), and we reject the hypothesis of independence. We estimated the portion of S0,basal which could not be accounted for by variability in ΔS0,purby regressing S0,basal onto ΔS0,pur, and this uncorrelated residual was ∼36% of S0,basal or 18% of the detergent control (Fig. 3 D). The correlation in the large data set was probably reduced by variability in the magnitude ofS0,basal and ΔS0,purover months of experiments, and the correlation between ΔS0,pur and S0,basalwas substantially higher (reaching values of 0.75) when smaller data sets (n < 20) collected over shorter periods of time were analyzed (data not shown). The correlation and regression analysis demonstrated that the processes underlyingS0,basal and ΔS0,purwere not independent, and a significant portion ofS0,basal could be predicted from ΔS0,pur. The activation of 4MαG is irreversible, and the hydrolysis of 4MαG by α-glucosidase could deplete the 4MαG concentration within a membrane-bound compartment if it is not replenished by a continuous entry of dye. The rate of entry of 4MαG is dependent on the permeability of the membrane to 4MαG and the concentration gradient for 4MαG across the membrane. This should produce an increase in the apparentKm of the α-glucosidase for 4MαG, with the magnitude of the increase inversely proportional to the permeability of the membrane to 4MαG. We measured the dependence ofS0,basal and ΔS0,pur on the concentration of 4MαG in cavitation-permeabilized cells to test this prediction. Both sets of data were fitted by Michaelis-Menten functions (Fig. 4). The ΔS0,pur data were fitted by a single component with an apparent Km near 80 μm (TableI). The S0,basaldata were fitted with two components, a dominant component with an apparent Km also near 80 μm and a very small, high affinity component. The apparent Km of ΔS0,pur and the dominant component ofS0,basal were increased ∼16-fold relative to the Km of the enzyme with detergent-permeabilized membranes (i.e. 5 μm, Fig. 1), and we conclude from the very similar shift in the apparent Kmvalues that S0,basal and ΔS0,pur are produced by the entry of dye via pathways with very similar permeabilities to 4MαG. The small, high affinity component in S0,basal might represent the activity of α-glucosidase released into the buffer, but the very small size of this component (<3% of total) provides additional evidence that nearly all of the activation of 4MαG in cavitation-permeabilized cells occurred within a membrane-bound compartment.Table IMembrane-dependent shift in apparent KmKmS0,maxμmDetergent-treated5.3 ± 0.2No detergentS0,basal0.7 ± 2.84.5 ± 6.179.4 ± 15.7152.5 ± 6.0ΔS0,pur83.1 ± 16.865.1 ± 6.9The dependence of S0 on 4MαG concentration was characterized by fitting the data sets with the functionS0 = S0,max/(1 +Km/C), where S0,max is the maximum slope, C is the concentration of 4MαG, andKm is the concentration of 4MαG which produces one-half S0,max. Parameter values are given ± S.E. of the parameter estimate. Open table in a new tab The dependence of S0 on 4MαG concentration was characterized by fitting the data sets with the functionS0 = S0,max/(1 +Km/C), where S0,max is the maximum slope, C is the concentration of 4MαG, andKm is the concentration of 4MαG which produces one-half S0,max. Parameter values are given ± S.E. of the parameter estimate. The puromycin-dependent activation of 4MαG is most easily accounted for by a puromycin-induced release of nascent polypeptide chains from the pores of translationally active translocons, with the empty pores providing additional pathways for the entry of 4MαG. Ribosomes can be dissociated from translocons by first releasing nascent polypeptide chains with puromycin, then increasing the ionic strength of the buffer above about 100 mm (23Adelman M.R. Sabatini D.D. Blobel G. J. Cell Biol. 1973; 56: 206-229Crossref PubMed Scopus (213) Google Scholar). Treatment with high salt has been shown to eliminate the permeation of translocon pores by ions (22Simon S.M. Blobel G. Cell. 1991; 65: 371-380Abstract Full Text PDF PubMed Scopus (485) Google Scholar). We examined the rate of activation of 4MαG in cavitation-permeabilized cells broken open in KG buffer containing 50, 140, 200, or 300 mm potassium glutamate. Increasing the potassium glutamate concentration decreased both S0,basal and ΔS0,pur in an identical, concentration-dependent manner (Fig.5). The concentration-dependent decreases in S0,basaland ΔS0,pur were fitted with a Hill function with very similar parameter values (Fig. 5 and TableII), a concentration midpoint near 150 mm and a Hill coefficient of 7.6. The high cooperativity was consistent with the breakage of multiple salt bonds, and the absence of a second component, as evident in the quality of the single component fit, indicated that all of the salt bonds had a similar sensitivity to the salt concentration. The concentration midpoint we observed was appropriate for the salt-dependent release of ribosomes (23Adelman M.R. Sabatini D.D. Blobel G. J. Cell Biol. 1973; 56: 206-229Crossref PubMed Scopus (213) Google Scholar). We conclude that the salt-sensitive step for ΔS0,pur is the salt-dependent release of ribosomes from translationally inactive, unoccupied translocons.Table IISalt-dependent inhibitionSmaxConst.Kih%%mS0,basal27.416.10.1517.6ΔS0,pur11.52.90.1537.6The slope data were normalized as a percentage of the detergent control and fitted with a Hill function of the following form:S(C) =Smax/[1 + (C/Ki)h] + Const., where Smax is the maximum relative slope,C is the potassium glutamate concentration,S(C) is the slope at C,Ki is the concentration at which there is half-maximal inhibition, h is the Hill coefficient, andConst. is the salt-insensitive activity. Open table in a new tab The slope data were normalized as a percentage of the detergent control and fitted with a Hill function of the following form:S(C) =Smax/[1 + (C/Ki)h] + Const., where Smax is the maximum relative slope,C is the potassium glutamate concentration,S(C) is the slope at C,Ki is the concentration at which there is half-maximal inhibition, h is the Hill coefficient, andConst. is the salt-insensitive activity. We surmised from the striking similarities in the inhibition of ΔS0,pur and S0,basal by high salt that the salt-sensitive release of ribosomes from unoccupied translocons was also the most likely mechanism underlying the reduction in S0,basal by high salt. This interpretation is especially attractive given recent evidence that the majority of large ribosomal subunits remain bound to the ER after the normal termination of protein synthesis (24Potter M.D. Nicchitta C.V. J. Biol. Chem. 2000; 275: 33828-33835Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 25Seiser R.M. Nicchitta C.V. J. Biol. Chem. 2000; 275: 33820-33827Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and this state would be equivalent to the ribosome-bound, unoccupied state of the translocon produced by treatment with puromycin (25Seiser R.M. Nicchitta C.V. J. Biol. Chem. 2000; 275: 33820-33827Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To strengthen this interpretation, we developed a quantitative model of the salt-sensitive and salt-insensitive components ofS0 in the absence (S0,basal) and the presence (S0,pur) of puromycin (Fig.6 and ). This model has three free parameters: f, the fraction of pores open in the absence of puromycin; x, the permeability of a blocked pore relative to an empty pore; and s, the fraction of empty pores from which ribosomes can be removed by high salt. We measuredS0,basal and S0,pur in a normal salt buffer (140 mm KG) and a high salt buffer (300 mm KG) (n = 14 experiments). The salt-sensitive component of S0,pur was then calculated and entered into the model, and the parameters f,x, and s were adjusted to optimize the fit toS0,basal and S0,pur under the normal and high salt conditions. As shown in Fig. 6 B, the fit was very good with parameter estimates of f = 0.738, x = 0.106, and s = 0.775. These parameter estimates were robust and independent of the starting values. Although this analysis indicated that 4MαG might permeate the blocked pores to a limited extent, it is clear from Fig. 6 B that permeation of empty pores is the primary pathway. We conclude from this analysis that the majority (74%) of the pores are permeable to 4MαG in the absence of puromycin, and this result is consistent with the persistent binding of ribosomes to translocons after termination of translation (24Potter M.D. Nicchitta C.V. J. Biol. Chem. 2000; 275: 33828-33835Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 25Seiser R.M. Nicchitta C.V. J. Biol. Chem. 2000; 275: 33820-33827Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We also note that the model predicted that 78% of the unoccupied pores are sensit" @default.
• W2017988657 created "2016-06-24" @default.
• W2017988657 creator A5049232345 @default.
• W2017988657 creator A5074393624 @default.
• W2017988657 date "2001-06-01" @default.
• W2017988657 modified "2023-10-10" @default.
• W2017988657 title "Translocon Pores in the Endoplasmic Reticulum Are Permeable to a Neutral, Polar Molecule" @default.
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