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Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling

82 Pages Posted: 12 May 2020 Sneak Peek Status: Under Review

See all articles by Evgeny Onishchenko

Evgeny Onishchenko

ETH Zürich - Institute of Biochemistry

Elad Noor

ETH Zürich - Institute of Molecular Systems Biology (IMSB)

Jonas Fischer

ETH Zürich - Institute of Biochemistry

Ludovic Gillet

ETH Zürich - Institute of Molecular Systems Biology (IMSB)

Matthias Wojtynek

ETH Zürich - Institute of Biochemistry

Pascal Vallotton

ETH Zürich - Institute of Biochemistry

Karsten Weis

ETH Zürich - Institute of Biochemistry

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Abstract

All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ~ 50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute-time-scale and then co-assemble from center to periphery in a ~1-hour-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily adaptable beyond the NPC, making it possible to analyze intracellular dynamics of a variety of multiprotein assemblies.

Keywords: maturation kinetics, macromolecular complex biogenesis, assembly order, nuclear pore complex, data independent acquisition, DIA mass spectrometry, INST-MFA, RITE, pulse-SILAC AP-MS, metabolic labeling

Suggested Citation

Onishchenko, Evgeny and Noor, Elad and Fischer, Jonas and Gillet, Ludovic and Wojtynek, Matthias and Vallotton, Pascal and Weis, Karsten, Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling. Available at SSRN: https://ssrn.com/abstract=3596618 or http://dx.doi.org/10.2139/ssrn.3596618
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Evgeny Onishchenko (Contact Author)

ETH Zürich - Institute of Biochemistry ( email )

Otto-Stern-Weg 3
Zürich, 8093
Sweden

Elad Noor

ETH Zürich - Institute of Molecular Systems Biology (IMSB) ( email )

Building HPT
Auguste-Piccard-Hof 1
Zürich, 8093
Switzerland

Jonas Fischer

ETH Zürich - Institute of Biochemistry

Otto-Stern-Weg 3
Zürich, 8093
Sweden

Ludovic Gillet

ETH Zürich - Institute of Molecular Systems Biology (IMSB) ( email )

Building HPT
Auguste-Piccard-Hof 1
Zürich, 8093
Switzerland

Matthias Wojtynek

ETH Zürich - Institute of Biochemistry ( email )

Otto-Stern-Weg 3
Zürich, 8093
Sweden

Pascal Vallotton

ETH Zürich - Institute of Biochemistry ( email )

Otto-Stern-Weg 3
Zürich, 8093
Sweden

Karsten Weis

ETH Zürich - Institute of Biochemistry ( email )

Otto-Stern-Weg 3
Zürich, 8093
Sweden

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