Nuclear Pore Membrane Proteins Self-Assemble into Nanopores

Jan 3, 2019 - Large multiprotein nanopores remain difficult to reconstitute in vitro, such as, for instance, the nuclear pore complex (NPC) that regul...
0 downloads 0 Views 553KB Size
Subscriber access provided by United Arab Emirates University | Libraries Deanship

Communication

Nuclear Pore Membrane Proteins Self-Assemble into Nanopores Radhakrishnan Panatala, Suncica Barbato, Toshiya Kozai, Jinghui Luo, Larisa E. Kapinos, and Roderick Y.H. Lim Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01179 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Nuclear Pore Membrane Proteins Self-Assemble into Nanopores Radhakrishnan Panatala1, Suncica Barbato1, Toshiya Kozai1, Jinghui Luo2, Larisa E. Kapinos1 and Roderick Y. H. Lim1,* 1Biozentrum 2Paul

and the Swiss Nanoscience Institute, University of Basel, 4056 Basel, Switzerland

Scherrer Institute, 5232 Villigen, Switzerland

author: [email protected] Phone: +41 61 207 2083; Fax: +41 61 207 2109 *Corresponding

ABSTRACT: Large multi-protein nanopores remain

difficult to reconstitute in vitro, such as for instance the nuclear pore complex (NPC) that regulates macromolecular transport between the nucleus and cytoplasm in cells. Here, we report that two NPC pore membrane proteins self-assemble into ~20 nm-diameter nanopores following in vitro reconstitution into lipid bilayers. Pore formation follows from the assembly of Pom121 and Ndc1 oligomers, which arrange into ring-like membrane structures that encircle aqueous, electrically conductive pores. This represents a key step towards reconstituting membrane-embedded NPC mimics for biological studies and biotechnological applications.

Membrane-embedded protein nanopores are important in several technologies such as DNA sequencing1,2 and protein biosensing3,4. These typically employ pore-forming toxins that comprise of β-barrel proteins (e.g., α-hemolysin) or α-helical barrels (e.g., ClyA) that present narrow channel diameters of 90% of all GPLs were stained with Nile Red, a lipid membrane stain. Among those, we followed the GFP signal on Ndc1 upon its standalone expression or co-expression with Pom121 (Figure 2c). This yielded Ndc1-eGFP signal intensities of ~30% for Pom121-Ndc1-GPLs and ~10% for Ndc1-GPLs. The control blank giantliposomes (GLs) gave null results (Figure 2c). At the nanoscale, high-speed atomic force microscopy (HS-AFM) revealed numerous pore-like perforations in the Pom121-Ndc1-PL membrane (Figure 3a) that were not present in blank liposome controls (Figure S2). Interestingly, each pore is enclosed by a ring-like structure above the membrane surface (Figure 3b). Each ring suggests that individual Pom121-Ndc1 sub-complexes assemble into a corral-like arrangement. Their average pore dimensions are 5.8 ± 1.4 nm, 19.5 ± 3.7 nm and 37.3 ± 6.1 nm for height (Hpore), inner diameter (i.d.) and outer diameter (o.d.), respectively (Figure 3b). Moreover, the ring height (Hrim) and thickness of the surrounding membrane (Hmem) as measured from the top of each ring and bottom of each pore to the membrane surface are 3.2 ± 1.4 nm and 2.5 ± 0.6 nm, respectively. Here, Hmem appears smaller than the expected lipid bilayer thickness37. This may be due to a limited penetration of the HS-AFM tip into the pore or that Pom121-Ndc1 pore formation might cause a local deformation (compression) of the lipid bilayer

surrounding it38. In addition, we sometimes observed features that resembled elongated pores (or “slits”) (Figure 3c) that were 107.7 ± 21.6 nm in length (Figure S3) but with similar Hpore, i.d. and o.d. values (Figure 3D). This suggests that the slits might form from conjoined pores. Figure 4: Ascending staircase-like conductance behavior indicates that the numbers of Pom121-Ndc1 pores are increasing over time. Each upward “step” signifies the emergence of a new pore but local fluctuations indicate stochastic behavior during protein insertion. As controls, blank liposomes, Pom121-PLs and Ndc1-PLs did not form pores.

Finally, we applied single channel electrical measurements39 to verify that the TM Nups formed aqueous pores that punctured the lipid membrane (Figure 4). In the absence of pores, such as in blank liposomes, the membrane exhibits a property of zero conductance. Similar results were obtained for Pom121- or Ndc1-only PLs, which did not show any evidence of pore formation. In marked contrast, Pom121-Ndc1-PLs exhibited an overall staircaselike behavior40 that ascended until the detection limit of the instrument was reached. Importantly, each upward “step” signifies the emergence of a new pore although local fluctuations suggest more complex behavior41 such as the formation, closure, and fusion of multiple pores. Moreover, each step can be used to estimate a theoretical pore size42 giving 3.8 ± 2.0 nm, which is in approximate agreement with our HS-AFM results. For completeness, pore formation did not occur when blank liposomes were added to Pom121-PLs and Ndc1-PLs respectively, nor during sequential additions of Ndc1-PLs to Pom121-PLs (Figure S4). However, the electrical signal destabilized when Pom121-PLs were introduced to Ndc1-PLs, which may suggest that Pom121 facilitates a rearrangement of Ndc1 in the membrane. Still, how the sequential addition of Pom121 and Ndc1 favors pore formation remains to be ascertained.

ACS Paragon Plus Environment

Page 4 of 8

Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

AUTHOR INFORMATION Corresponding Author *[email protected]

Funding Sources

No competing financial interests have been declared.

To summarize, our work presents Pom121-Ndc1 pores in lipid membranes. Their diameters are smaller than those of NPCs, as NPCs are formed by fusing the inner and outer nuclear membranes26 whereas Pom121-Ndc1 pores are embedded in lipid bilayers (Figure S5). More in-depth studies will be required to resolve the structure and orientation of each protein, as well as how they self-assemble and from which side of the PL membrane the ring structures emanate i.e., luminal or external. Furthermore, the Pom121 FG domain binds Kapβ1 and the RanGDP importer nuclear transport factor 2 (NTF2) with a binding affinity of 71 ± 15.4 nM and 2.77 ± 0.31 µM, respectively (Figure S6). Hence, Pom121-Ndc1 pores may be able to facilitate the selective transport of NTRs to serve as NPC mimics. We further envisage that the Pom121-Ndc1 pores may be used as templates to build on other NPC components that interact with Pom121 and Ndc128. This opens up new strategies to de novo engineer NPC-based nanopores for biological studies and diverse biotechnological applications43.

R.P. and S.B. are supported by the National Centre of Competence in Research in Molecular Systems Engineering. T.K. is supported by a Swiss Nanoscience Institute Fellowship. Further support to R.Y.H.L. is provided by a Swiss National Science Foundation grant no. 31003A_170041.

ACKNOWLEDGMENT We thank Thomas K.C. Bock and Alexander Schmidt from the Proteomics Core Facility, Janine Bögli from the FACS Facility and Yusuke Sakiyama for advice and technical assistance with HS-AFM.

REFERENCES (1) Venkatesan, B. M., and Bashir, R. (2011) Nanopore sensors for nucleic acid analysis. Nat. Nanotechnol. 6, 615–624. (2) Clarke, J., Wu, H.-C., Jayasinghe, L., Patel, A., Reid, S., and Bayley, H. (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265–270. (3) Robertson, J. W. F., and Reiner, J. E. (2018) The utility of nanopore technology for protein and peptide sensing. Proteomics 18, e1800026. (4) Huang, G., Willems, K., Soskine, M., Wloka, C., and Maglia, G. (2017) Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores. Nat Commun 8, 935. (5) Willems, K., Van Meervelt, V., Wloka, C., and Maglia, G. (2017) Single-molecule nanopore enzymology. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 372.

ASSOCIATED CONTENT Supporting Information. Supplementary Figures for Figures 1, 3 and 4, and Supplementary Figures S5 and S6 with their respective figure legends. Excel File showing mass spectrometry data and analysis. Materials and Methods.

(6) Howorka, S. (2017) Building membrane nanopores. Nat. Nanotechnol. 12, 619–630. (7) Ayub, M., and Bayley, H. (2016) Engineered transmembrane pores. Curr. Opin. Chem. Biol. 34, 117–126. (8) Soskine, M., Biesemans, A., De Maeyer, M., and Maglia, G. (2013) Tuning the size and properties of ClyA nanopores assisted by directed evolution. J. Am. Chem. Soc. 135, 13456–13463.

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(9) Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, T. J., Montemagno, C., and Guo, P. (2009) Translocation of doublestranded DNA through membrane-adapted phi29 motor protein nanopores. Nat. Nanotechnol. 4, 765–772. (10) Tweten, R. K. (2005) Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins. Infect. Immun. 73, 6199–6209. (11) Wente, S. R., and Rout, M. P. (2010) The nuclear pore complex and nuclear transport. Cold Spring Harb. Perspect. Biol. 2, a000562. (12) von Appen, A., Kosinski, J., Sparks, L., Ori, A., DiGuilio, A. L., Vollmer, B., Mackmull, M.-T., Banterle, N., Parca, L., Kastritis, P., Buczak, K., Mosalaganti, S., Hagen, W., Andres-Pons, A., Lemke, E. A., Bork, P., Antonin, W., Glavy, J. S., Bui, K. H., and Beck, M. (2015) In situ structural analysis of the human nuclear pore complex. Nature 526, 140–143. (13) Kim, S. J., Fernandez-Martinez, J., Nudelman, I., Shi, Y., Zhang, W., Raveh, B., Herricks, T., Slaughter, B. D., Hogan, J. A., Upla, P., Chemmama, I. E., Pellarin, R., Echeverria, I., Shivaraju, M., Chaudhury, A. S., Wang, J., Williams, R., Unruh, J. R., Greenberg, C. H., Jacobs, E. Y., Yu, Z., de la Cruz, M. J., Mironska, R., Stokes, D. L., Aitchison, J. D., Jarrold, M. F., Gerton, J. L., Ludtke, S. J., Akey, C. W., Chait, B. T., Sali, A., and Rout, M. P. (2018) Integrative structure and functional anatomy of a nuclear pore complex. Nature 555, 475–482. (14) Grossman, E., Medalia, O., and Zwerger, M. (2012) Functional architecture of the nuclear pore complex. Annu. Rev. Biophys. 41, 557–584. (15) Sakiyama, Y., Mazur, A., Kapinos, L. E., and Lim, R. Y. H. (2016) Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nat. Nanotechnol. 11, 719–723. (16) Bayliss, R., Littlewood, T., and Stewart, M. (2000) Structural Basis for the Interaction between FxFG Nucleoporin Repeats and Importin-β in Nuclear Trafficking. Cell 102, 99–108. (17) Kapinos, L. E., Huang, B., Rencurel, C., and Lim, R. Y. H. (2017) Karyopherins regulate nuclear pore complex barrier and transport function. J. Cell Biol. 216, 3609–3624. (18) Timney, B. L., Raveh, B., Mironska, R., Trivedi, J. M., Kim, S. J., Russel, D., Wente, S. R., Sali, A., and Rout, M. P. (2016) Simple rules for passive diffusion through the nuclear pore complex. J. Cell Biol. 215, 57–76. (19) Popken, P., Ghavami, A., Onck, P. R., Poolman, B., and Veenhoff, L. M. (2015) Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex. Mol. Biol. Cell 26, 1386– 1394. (20) Jovanovic-Talisman, T., Tetenbaum-Novatt, J., McKenney, A. S., Zilman, A., Peters, R., Rout, M. P., and Chait, B. T. (2009) Artificial nanopores that mimic the transport selectivity of the nuclear pore complex. Nature 457, 1023–1027. (21) Kowalczyk, S. W., Kapinos, L., Blosser, T. R., Magalhães, T., van Nies, P., Lim, R. Y. H., and Dekker, C. (2011) Single-molecule transport across an individual biomimetic nuclear pore complex. Nat. Nanotechnol. 6, 433–438. (22) Fisher, P. D. E., Shen, Q., Akpinar, B., Davis, L. K., Chung, K. K. H., Baddeley, D., Šarić, A., Melia, T. J., Hoogenboom, B. W., Lin, C., and

Lusk, C. P. (2018) A Programmable DNA Origami Platform for Organizing Intrinsically Disordered Nucleoporins within Nanopore Confinement. ACS Nano 12, 1508–1518. (23) Ketterer, P., Ananth, A. N., Laman Trip, D. S., Mishra, A., Bertosin, E., Ganji, M., van der Torre, J., Onck, P., Dietz, H., and Dekker, C. (2018) DNA origami scaffold for studying intrinsically disordered proteins of the nuclear pore complex. Nat Commun 9, 902. (24) Alber, F., Dokudovskaya, S., Veenhoff, L. M., Zhang, W., Kipper, J., Devos, D., Suprapto, A., Karni-Schmidt, O., Williams, R., Chait, B. T., Sali, A., and Rout, M. P. (2007) The molecular architecture of the nuclear pore complex. Nature 450, 695–701. (25) Bui, K. H., von Appen, A., DiGuilio, A. L., Ori, A., Sparks, L., Mackmull, M.-T., Bock, T., Hagen, W., Andrés-Pons, A., Glavy, J. S., and Beck, M. (2013) Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155, 1233–1243. (26) Rothballer, A., and Kutay, U. (2013) Poring over pores: nuclear pore complex insertion into the nuclear envelope. Trends Biochem. Sci. 38, 292–301. (27) Mansfeld, J., Güttinger, S., Hawryluk-Gara, L. A., Panté, N., Mall, M., Galy, V., Haselmann, U., Mühlhäusser, P., Wozniak, R. W., Mattaj, I. W., Kutay, U., and Antonin, W. (2006) The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells. Mol. Cell 22, 93–103. (28) Mitchell, J. M., Mansfeld, J., Capitanio, J., Kutay, U., and Wozniak, R. W. (2010) Pom121 links two essential subcomplexes of the nuclear pore complex core to the membrane. J. Cell Biol. 191, 505–521. (29) Hallberg, E., Wozniak, R. W., and Blobel, G. (1993) An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region. J. Cell Biol. 122, 513– 521. (30) Stavru, F., Hülsmann, B. B., Spang, A., Hartmann, E., Cordes, V. C., and Görlich, D. (2006) NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes. J. Cell Biol. 173, 509–519. (31) Goren, M. A., Nozawa, A., Makino, S., Wrobel, R. L., and Fox, B. G. (2009) Cell-free translation of integral membrane proteins into unilamelar liposomes. Meth. Enzymol. 463, 647–673. (32) Kol, M., Panatala, R., Nordmann, M., Swart, L., van Suijlekom, L., Cabukusta, B., Hilderink, A., Grabietz, T., Mina, J. G. M., Somerharju, P., Korneev, S., Tafesse, F. G., and Holthuis, J. C. M. (2017) Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases. J. Lipid Res. 58, 962–973. (33) Labokha, A. A., Gradmann, S., Frey, S., Hülsmann, B. B., Urlaub, H., Baldus, M., and Görlich, D. (2013) Systematic analysis of barrierforming FG hydrogels from Xenopus nuclear pore complexes. EMBO J. 32, 204–218. (34) Silva, J. C., Gorenstein, M. V., Li, G.-Z., Vissers, J. P. C., and Geromanos, S. J. (2006) Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. Mol. Cell Proteomics 5, 144–156. (35) Ahrné, E., Molzahn, L., Glatter, T., and Schmidt, A. (2013) Critical

ACS Paragon Plus Environment

Page 6 of 8

Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry assessment of proteome-wide label-free absolute abundance estimation strategies. Proteomics 13, 2567–2578. (36) Ori, A., Banterle, N., Iskar, M., Andrés-Pons, A., Escher, C., Khanh Bui, H., Sparks, L., Solis-Mezarino, V., Rinner, O., Bork, P., Lemke, E. A., and Beck, M. (2013) Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol. Syst. Biol. 9, 648. (37) Nagle, J. F., and Tristram-Nagle, S. (2000) Structure of lipid bilayers. Biochim. Biophys. Acta 1469, 159–195. (38) Gilbert, R. J. C. (2016) Protein-lipid interactions and nonlamellar lipidic structures in membrane pore formation and membrane fusion. Biochim. Biophys. Acta 1858, 487–499. (39) Bayley, H., and Martin, C. R. (2000) Resistive-Pulse SensingFrom Microbes to Molecules. Chem. Rev. 100, 2575–2594. (40) Bode, D. C., Baker, M. D., and Viles, J. H. (2017) Ion Channel Formation by Amyloid-β42 Oligomers but Not Amyloid-β40 in Cellular Membranes. J. Biol. Chem. 292, 1404–1413. (41) Sekiya, Y., Sakashita, S., Shimizu, K., Usui, K., and Kawano, R. (2018) Channel current analysis estimates the pore-formation and the penetration of transmembrane peptides. Analyst 143, 3540– 3543. (42) Cruickshank, C. C., Minchin, R. F., Le Dain, A. C., and Martinac, B. (1997) Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli. Biophys. J. 73, 1925–1931. (43) Vujica, S., Zelmer, C., Panatala, R., and Lim, R. Y. H. (2016) Nucleocytoplasmic transport: A paradigm for molecular logistics in artificial systems. Chimia (Aarau). 70, 413–417.

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only

ACS Paragon Plus Environment

Page 8 of 8