Skip to main content
SpringerLink
Log in

Constitutive, translation-independent opening of the protein-conducting channel in the endoplasmic reticulum

  • Signaling and Cell Physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Secretory and membrane proteins are translocated into the endoplasmic reticulum (ER) through a translocon assembled as a tetramer of Sec61 protein-conducting channels (PCC). How the opening of the PCCs in the tetramer is regulated through the protein translocation cycle is poorly understood. In this study, the permeability of PCCs in native ER membranes to small molecules was measured using fluorescence and electrophysiological techniques. Although the PCCs were closed at 4°C, they were constitutively open at physiological temperatures in the absence of protein translation or a bound ribosome. The open PCCs occurred in clusters that are likely to correspond to the simultaneous opening of three or four PCCs in a translocon. The binding of 60S subunits to a ribosome-free membrane increased the number of open PCCs but did not increase the single-channel conductance. The translation-independent, constitutive opening of Sec61 PCCs provides new insight into the role of the translocon in the transport of small molecules across the ER membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Adelman MR, Sabatini DD, Blobel G (1973) Ribosome–membrane interaction. Nondestructive disassembly of rat liver rough microsomes into ribosomal and membranous components. J Cell Biol 56:206–229

    Article  PubMed  CAS  Google Scholar 

  2. Beckmann R, Spahn CM, Eswar N, Helmers J, Penczek PA, Sali A, Frank J, Blobel G (2001) Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107:361–372

    Article  PubMed  CAS  Google Scholar 

  3. Borgese D, Blobel G, Sabatini DD (1973) In vitro exchange of ribosomal subunits between free and membrane-bound ribosomes. J Mol Biol 74:415–438

    Article  PubMed  CAS  Google Scholar 

  4. Boyd IA, Martin AR (1956) The end-plate potential in mammalian muscle. J physiol 132:74–91

    PubMed  CAS  Google Scholar 

  5. Cannon KS, Or E, Clemons WM Jr, Shibata Y, Rapoport TA (2005) Disulfide bridge formation between SecY and a translocating polypeptide localizes the translocation pore to the center of SecY. J Cell Biol 169:219–225

    Article  PubMed  CAS  Google Scholar 

  6. Clark AG, Murray D, Ashley RH (1997) Single-channel properties of a rat brain endoplasmic reticulum anion channel. Biophys J 73:168–178

    Article  PubMed  CAS  Google Scholar 

  7. Criado M, Keller BU (1987) A membrane fusion strategy for single-channel recordings of membranes usually non-accessible to patch-clamp pipette electrodes. FEBS letters 224:172–176

    Article  PubMed  CAS  Google Scholar 

  8. Flourakis M, Van Coppenolle F, Lehen’kyi V, Beck B, Skryma R, Prevarskaya N (2006) Passive calcium leak via translocon is a first step for iPLA2-pathway regulated store operated channels activation. Faseb J 20:1215–1217

    Article  PubMed  CAS  Google Scholar 

  9. Giunti R, Gamberucci A, Fulceri R, Banhegyi G, Benedetti A (2007) Both translocon and a cation channel are involved in the passive Ca(2+) leak from the endoplasmic reticulum: a mechanistic study on rat liver microsomes. Arch Biochem Biophys 462:115–121

    Article  PubMed  CAS  Google Scholar 

  10. Gumbart J, Schulten K (2006) Molecular dynamics studies of the archaeal translocon. Biophys J 90:2356–2367

    Article  PubMed  CAS  Google Scholar 

  11. Haider S, Hall BA, Sansom MS (2006) Simulations of a protein translocation pore: SecY. Biochemistry 45:13018–13024

    Article  PubMed  CAS  Google Scholar 

  12. Hamilton MG, Ruth ME (1969) The dissociation of rat liver ribosomes by ethylenediaminetetraacetic acid; molecular weights, chemical composition, and buoyant densities of the subunits. Biochemistry 8:851–856

    Article  PubMed  CAS  Google Scholar 

  13. Hamman BD, Hendershot LM, Johnson AE (1998) BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation. Cell 92:747–758

    Article  PubMed  CAS  Google Scholar 

  14. Hanein D, Matlack KE, Jungnickel B, Plath K, Kalies KU, Miller KR, Rapoport TA, Akey CW (1996) Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell 87:721–732

    Article  PubMed  CAS  Google Scholar 

  15. Heritage D, Wonderlin WF (2001) Translocon pores in the endoplasmic reticulum are permeable to a neutral, polar molecule. J Biol Chem 276:22655–22662

    Article  PubMed  CAS  Google Scholar 

  16. Hofer AM, Curci S, Machen TE, Schulz I (1996) ATP regulates calcium leak from agonist-sensitive internal calcium stores. Faseb J 10:302–308

    PubMed  CAS  Google Scholar 

  17. Hofer AM, Schlue WR, Curci S, Machen TE (1995) Spatial distribution and quantitation of free luminal [Ca] within the InsP3-sensitive internal store of individual BHK-21 cells: ion dependence of InsP3-induced Ca release and reloading. Faseb J 9:788–798

    PubMed  CAS  Google Scholar 

  18. Johnson AE, van Waes MA (1999) The translocon: a dynamic gateway at the ER membrane. Annu Rev Cell Dev Biol 15:799–842

    Article  PubMed  Google Scholar 

  19. Kalies KU, Gorlich D, Rapoport TA (1994) Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex. J Cell Biol 126:925–934

    Article  PubMed  CAS  Google Scholar 

  20. Le Gall S, Neuhof A, Rapoport T (2004) The endoplasmic reticulum membrane is permeable to small molecules. Mol Biol Cell 15:447–455

    Article  PubMed  CAS  Google Scholar 

  21. Li W, Schulman S, Boyd D, Erlandson K, Beckwith J, Rapoport TA (2007) The plug domain of the SecY protein stabilizes the closed state of the translocation channel and maintains a membrane seal. Mol Cell 26:511–521

    Article  PubMed  CAS  Google Scholar 

  22. Lizak B, Csala M, Benedetti A, Banhegyi G (2008) The translocon and the non-specific transport of small molecules in the endoplasmic reticulum (review). Mol Memb Biol 25:95–101

    Article  CAS  Google Scholar 

  23. Lizak B, Czegle I, Csala M, Benedetti A, Mandl J, Banhegyi G (2006) Translocon pores in the endoplasmic reticulum are permeable to small anions. Am J physiol 291:C511–517

    Article  CAS  Google Scholar 

  24. Lomax RB, Camello C, Van Coppenolle F, Petersen OH, Tepikin AV (2002) Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons. J Biol Chem 277:26479–26485

    Article  PubMed  CAS  Google Scholar 

  25. Marcolongo P, Fulceri R, Giunti R, Burchell A, Benedetti A (1996) Permeability of liver microsomal membranes to glucose. Biochem Biophys Res Commun 219:916–922

    Article  PubMed  CAS  Google Scholar 

  26. Meissner G, Allen R (1981) Evidence for two types of rat liver microsomes with differing permeability to glucose and other small molecules. J Biol Chem 256:6413–6422

    PubMed  CAS  Google Scholar 

  27. Menetret JF, Hegde RS, Heinrich SU, Chandramouli P, Ludtke SJ, Rapoport TA, Akey CW (2005) Architecture of the ribosome–channel complex derived from native membranes. J Mol Biol 348:445–457

    Article  PubMed  CAS  Google Scholar 

  28. Morgan DG, Menetret JF, Neuhof A, Rapoport TA, Akey CW (2002) Structure of the mammalian ribosome–channel complex at 17A resolution. J Mol biol 324:871–886

    Article  PubMed  CAS  Google Scholar 

  29. Morier N, Sauve R (1994) Analysis of a novel double-barreled anion channel from rat liver rough endoplasmic reticulum. Biophys J 67:590–602

    Article  PubMed  CAS  Google Scholar 

  30. Ong HL, Liu X, Sharma A, Hegde RS, Ambudkar IS (2007) Intracellular Ca(2+) release via the ER translocon activates store-operated calcium entry. Pflugers Arch 453:797–808

    Article  PubMed  CAS  Google Scholar 

  31. Osborne AR, Rapoport TA (2007) Protein translocation is mediated by oligomers of the SecY complex with one SecY copy forming the channel. Cell 129:97–110

    Article  PubMed  CAS  Google Scholar 

  32. Palmer AE, Jin C, Reed JC, Tsien RY (2004) Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci USA 101:17404–17409

    Article  PubMed  CAS  Google Scholar 

  33. Prinz A, Behrens C, Rapoport TA, Hartmann E, Kalies KU (2000) Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA. Embo J 19:1900–1906

    Article  PubMed  CAS  Google Scholar 

  34. Raden D, Song W, Gilmore R (2000) Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex. J Cell Biol 150:53–64

    Article  PubMed  CAS  Google Scholar 

  35. Rapoport TA (2007) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450:663–669

    Article  PubMed  CAS  Google Scholar 

  36. Roy A, Wonderlin WF (2002) The permeability of the endoplasmic reticulum is dynamically coupled to protein synthesis. J Biol Chem 278:4397–4403

    Article  PubMed  CAS  Google Scholar 

  37. Saparov SM, Erlandson K, Cannon K, Schaletzky J, Schulman S, Rapoport TA, Pohl P (2007) Determining the conductance of the SecY protein translocation channel for small molecules. Mol Cell 26:501–509

    Article  PubMed  CAS  Google Scholar 

  38. Schaletzky J, Rapoport TA (2006) Ribosome binding to and dissociation from translocation sites of the endoplasmic reticulum membrane. Mol Biol Cell 17:3860–3869

    Article  PubMed  CAS  Google Scholar 

  39. Schultz SG, Solomon AK (1961) Determination of the effective hydrodynamic radii of small molecules by viscometry. J Gen Physiolx 44:1189–1199

    Article  CAS  Google Scholar 

  40. Seiser RM, Nicchitta CV (2000) The fate of membrane-bound ribosomes following the termination of protein synthesis. J Biol Chem 275:33820–33827

    Article  PubMed  CAS  Google Scholar 

  41. Simon SM, Blobel G (1991) A protein-conducting channel in the endoplasmic reticulum. Cell 65:371–380

    Article  PubMed  CAS  Google Scholar 

  42. Simon SM, Blobel G (1992) Signal peptides open protein-conducting channels in E. coli. Cell 69:677–684

    Article  PubMed  CAS  Google Scholar 

  43. Simon SM, Blobel G, Zimmerberg J (1989) Large aqueous channels in membrane vesicles derived from the rough endoplasmic reticulum of canine pancreas or the plasma membrane of Escherichia coli. Proc Natl Acad Sci USA 86:6176–6180

    Article  PubMed  CAS  Google Scholar 

  44. Tam PC, Maillard AP, Chan KK, Duong F (2005) Investigating the SecY plug movement at the SecYEG translocation channel. EMBO J 24:3380–3388

    Article  PubMed  CAS  Google Scholar 

  45. Tani K, Tokuda H, Mizushima S (1990) Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization. J Biol Chem 265:17341–17347

    PubMed  CAS  Google Scholar 

  46. Tian P, Andricioaei I (2006) Size, motion, and function of the SecY translocon revealed by molecular dynamics simulations with virtual probes. Biophys J 90:2718–2730

    Article  PubMed  CAS  Google Scholar 

  47. Van den Berg B, Clemons WM Jr, Collinson I, Modis Y, Hartmann E, Harrison SC, Rapoport TA (2004) X-ray structure of a protein-conducting channel. Nature 427:36–44

    Article  PubMed  CAS  Google Scholar 

  48. Walter P, Blobel G (1983) Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol 96:84–93

    Article  PubMed  CAS  Google Scholar 

  49. Wirth A, Jung M, Bies C, Frien M, Tyedmers J, Zimmermann R, Wagner R (2003) The Sec61p complex is a dynamic precursor activated channel. Mol Cell 12:261–268

    Article  PubMed  CAS  Google Scholar 

  50. Wool IG (1979) The structure and function of eukaryotic ribosomes. Ann Rev Boichem 48:719–754

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The author would like to thank Carol Deutsch, Ramanujan Hegde, and Andrew Shiemke for helpful comments on a draft of this paper.

Author information

Authors and Affiliations

  1. Department of Biochemistry, West Virginia University, P.O. Box 9142, Morgantown, WV, 26506, USA

    William F. Wonderlin

Authors
  1. William F. Wonderlin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William F. Wonderlin.

Electronic supplementary material

Below is the link to the electronic supplementary material

Fig. S1

Analysis by velocity sedimentation on 10–35% sucrose gradients (6 h at 35,000 rpm in SW40 rotor) of ribosomal RNA (rRNA) in samples of 60S and 40S subunits treated with 0.5% SDS at 37°C for 5 min. The pooled 60S and 40S fractions contained 28S and 18S rRNA, whereas the 60S peak used in the permeability assays contained only 28S rRNA. (DOC 32.0 KB)

Fig. S2

The permeability of CHO-K1 cells to 4 MαG was compared in a nominally calcium-free solution and in a solution containing 500 nM free calcium plus 2 mM ATP. The nominally calcium-free solution included 140 mM K-glutamate, 10 mM HEPES, 4 mM EGTA, and 3.25 mM MgCl2, pH 7.2. The buffered calcium/ATP solution included 4 mM EGTA, 3 mM CaCl2, 4.65 mM MgCl2, 2 mM Na-ATP, 10 mM HEPES, pH 7.2. The free magnesium concentration in both solutions was 2.5 mM. The concentrations of free calcium and free magnesium where calculated using WEBMAXC software (Stanford Univ.). (Average of three assays ± SEM). (DOC 46.5 KB)

Fig. S3

Distribution of the number of channels per patch for each of the primary treatments. The data in the histogram in Fig. 6a were pooled from recordings using microsomes fused after a variety of treatments. The most common treatments were (1) EDTA and high K (500 mM KCl), (2) puromycin and high K, or (3) high K alone. These treatments were combined with the variable addition of 60S subunits to the bath or to the pipet solution. The data for five conditions are shown in this figure. The sample size is largest for microsomes stripped with EDTA and high K, ±60S subunits, and these data reveal most clearly that the tendency to cluster was not dependent on the presence of 60S subunits, although there were more channels per patch in the presence of 60S subunits. Sixty-six of the 79 data included in the histogram in Fig. 6a are plotted by treatment group. E = EDTA. HK = 500 mM KCl. PUR = 100 μM puromycin. 60 = 60S subunits. The horizontal bar is the mean for each treatment. Dotted lines indicate the positions of the troughs in the multi-modal distribution shown in Fig. 6a. Clustering is evident between 0.5–1, 3–4, and >8 channels per patch. These data support our conclusion that clustering was a fundamental property associated with physiologically active channels, regardless of how the microsomes were treated. (DOC 55.0 KB)

Fig. S4

Pig pancreatic microsomes were incubated with 20 mM EDTA or 500 mM K-acetate/1 mM PUR for 20 min. The samples were diluted 9-fold with 2.22 M sucrose/0.5 M K-acetate and overlaid with 1.5 M sucrose/0.5 M K-acetate and 0.5 M K-acetate solutions. The step gradient was centrifuged at 100,000 rpm in a TL100.2 rotor for 60 min. The interface between the 1.5 M sucrose and 0.5 M K-acetate solutions was collected, diluted, and pelleted. The decrease in the permeability to 4 MαG produced by the treatment with EDTA or high salt/puromycin (comparison marked by bracket) was not significantly different (two-sided t test, α = 0.05). Both treatments also eliminated the stimulatory effect of puromycin added to the assay buffer (average of five assays ± SEM). (DOC 51.0 KB)

Fig. S5

Pig microsomes were stripped with 13 mM EDTA in 140KG, then diluted 1:1 with 140KG buffer containing digitonin (80 μg/ml). After 5 min, the sample was diluted 1:50 with control 140KG and used in the 4 MαG assay (average of four assays ± SEM). (DOC 40.5 KB)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wonderlin, W.F. Constitutive, translation-independent opening of the protein-conducting channel in the endoplasmic reticulum. Pflugers Arch - Eur J Physiol 457, 917–930 (2009). https://doi.org/10.1007/s00424-008-0545-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-008-0545-y

Keywords

Search

Navigation