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. 2004 Feb;15(2):447-55.
doi: 10.1091/mbc.e03-05-0325. Epub 2003 Nov 14.

The endoplasmic reticulum membrane is permeable to small molecules

Sylvie Le Gall  1 , Andrea Neuhof, Tom Rapoport
Affiliations

The endoplasmic reticulum membrane is permeable to small molecules

Sylvie Le Gall et al. Mol Biol Cell. 2004 Feb.

Abstract

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of approximately 5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.

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Figures

Figure 1.
Figure 1.
Ribosome-stripped ER membranes are permeable to small molecules. (A) Ribosomes carrying a radiolabeled fragment of ppαF (86mer) with a single cysteine at position 56 were incubated with ribosome-stripped microsomes (PK-RM). After treatment with puromycin to release the nascent chains into the ER lumen, the samples were incubated with 0.1 mM MB or 0.075 mM MPB for 10 min on ice, either in the absence of detergent, or in the presence of 0.5% TX-100 or 0.1% digitonin, as indicated. One-half of the samples was analyzed after precipitation with TCA (total), the other was subjected to incubation with streptavidin beads, and the bound (beads) and unbound (supernatant) fractions were analyzed. All samples were analyzed by SDS-PAGE and autoradiography. The stars indicate molecules with one to three carbohydrate chains. (B) Full-length ppαF with a single cysteine at position 56 was imported into untreated RMs. Membranes were pelleted before the modification reaction was carried out. The samples were then treated for 10 min on ice with the indicated concentrations of MB in the absence or presence of 0.5% Triton X-100. One half of the sample was analyzed directly (total), the other was subjected to incubation with streptavidin beads and the bound material was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with one to three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.
Figure 2.
Figure 2.
Rough microsomes are permeable to a modification reagent. Radiolabeled full-length ppαF was translocated into rough microsomes. The membranes were isolated and incubated with different concentrations of sulfo-NHS-biotin, an amino group-modifying reagent, for 10 min on ice or at 30°C. Where indicated, 0.5% TX-100 was present during the modification reaction. One portion of the samples was analyzed directly (total), the other was incubated with streptavidin beads and the bound fraction was analyzed (beads). All samples were subjected to SDS-PAGE and autoradiography. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. Molecular masses (in kilodaltons) are indicated on the right side.
Figure 3.
Figure 3.
Time course of modification of ppαF. (A) Radiolabeled full-length ppαF with a cysteine at position 56 was translocated into rough microsomes. The samples were centrifuged resulting in P and S fractions. The membranes were incubated with 50 μM MB for different time periods on ice. One-half of the samples was analyzed after TCA precipitation; the other was incubated with streptavidin beads and the bound material was analyzed. ppαF, gppαF, pro-α-factor without or with three carbohydrate chains, respectively. (B) Quantification of the experiment in A.
Figure 4.
Figure 4.
An endogenous luminal ER protein is accessible to a modification reagent. (A) Ribosome-stripped microsomes (PK-RM) were incubated with 100 μM MB on ice for different time periods in the absence or presence of 0.5% TX-100. The samples were immunoprecipitated with antibodies to PDI, subjected to SDS-PAGE, transferred onto a nitrocellulose filter, and incubated with peroxidase-coupled streptavidin. (B) As in A, but immunoprecipitation with antibodies to Sec61α.
Figure 5.
Figure 5.
Differential permeabilization of plasma and ER membranes. HeLa cells (2 × 106) were permeabilized with SLO or digitonin (Dig; 0.04 or 0.1%). The cell remnants were pelleted in a Microfuge, and S and P fractions, corresponding to 5 × 104 cells, were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.
Figure 6.
Figure 6.
A small molecule passes into the ER but not into lysosomes or the trans-Golgi. HeLa cells (2 × 106) were permeabilized with SLO, 0.04% digitonin (Dig), or 0.1% digitonin as indicated. Then 0.5 mM sulfo-NHS-LC-biotin was added for 30 min on ice. After solubilization in detergent, cell lysates were incubated with streptavidin agarose beads, and the bound material was analyzed by SDS-PAGE and immunoblotting with antibodies to various proteins. Hex B, hexosaminidase B; Cath D, cathepsin D; βGT1; β-1,4-galactosyl transferase. For βGT1, twice as much sample was used. Cell lysate corresponding to 10% of the input material was loaded as a control (lane 1).
Figure 7.
Figure 7.
The ER membrane does not constitute a significant barrier to small molecules. HeLa cells were permeabilized with 0.04% digitonin (A) or SLO (B) and incubated with 0.5 mM sulfo-NHS-LC-biotin for different periods of time. Biotinylated proteins were recovered after incubation with streptavidin beads and analyzed by SDS-PAGE and immunoblotting with antibodies to different proteins. The bands were quantitated using a Fujix PhosporIimager and the software Image Gauge 3.0.
Figure 8.
Figure 8.
Modification of metabolically labeled ER proteins occurs only upon cell permeabilization. (A) HeLa cells expressing HA-tagged MHC class I heavy chains with lysine residues only in the luminal domain (HA-heavy chain; K-R) were labeled with [35S]methionine and -cysteine. The cells were permeabilized with digitonin (Dig), as indicated, and treated with the amino group modifying reagent sulfo-NHS-biotin. The heavy chains were immunoprecipitated with HA antibodies. 20% of the immunoprecipitated material was analyzed directly by SDS-PAGE (lanes 1-4), and 80% was incubated with streptavidin beads to detect biotinylated material (lanes 5-8). Where indicated, one-half of each sample was treated with Endo H. All samples were subjected to SDS-PAGE followed by autoradiography. (B) Hybridoma B cells expressing IgG were analyzed as in A, except that the labeled IgG was collected with protein A/G-Sepharose.
Figure 9.
Figure 9.
A reagent of about 5 kDa cannot pass the ER membrane. HeLa cells (2 × 106) were permeabilized with 0.04% digitonin or lysed in 1% Triton X-100 as indicated. Modification was carried out for 30 min at 4°C with 1.5 mM mPEG, an SH-reagent of ∼5 kDa. Where indicated, DTT was present during the reaction. Samples corresponding to 5 × 104 untreated (T) or mPEG-modified cells were analyzed by SDS-PAGE on a 7.5-17.5% urea gel, followed by immunoblotting with antibodies to GRP94 or Sec61β. xSec 61β and xGRP94 indicate the positions of the modified proteins.
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