A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

TY - JOUR

T1 - A nanobody suite for yeast scaffold nucleoporins provides details of the nuclear pore complex structure

AU - Nordeen, Sarah A.

AU - Andersen, Kasper R.

AU - Knockenhauer, Kevin E.

AU - Ingram, Jessica R.

AU - Ploegh, Hidde L.

AU - Schwartz, Thomas U.

N1 - Funding Information: We thank Nina C. Leksa and Xuanzong Guo for their initial work on the Nup120 structure and members of the Schwartz lab for discussions on the experiments and manuscript. We thank Karsten Weis for advice on fluorescence microscopy and experiments in yeast. The research was supported by the US NIH under grant number R01GM77537 (K.E.K., K.A., and T.U.S.) and T32GM007287 (S.A.N.). J.R.I. and H.P. were supported by an NIH Pioneer award. The X-ray crystallography work was conducted at the APS NE-CAT beamlines, which are supported by NIH award GM103403. Use of the APS is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Publisher Copyright: © 2020, The Author(s).

PY - 2020/12

Y1 - 2020/12

N2 - Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

AB - Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we here develop a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae detect accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.

UR - https://www.scopus.com/pages/publications/85097016888

U2 - 10.1038/s41467-020-19884-6

DO - 10.1038/s41467-020-19884-6

M3 - Journal article

C2 - 33268786

AN - SCOPUS:85097016888

SN - 2041-1723

VL - 11

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 6179

ER -