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Protein Dynamics in Living Cells

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Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling.

Evgeny Onischenko¹, Elad Noor², Jonas S Fischer³

¹Department of Biological Sciences, University of Bergen, Bergen 5020, Norway; Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich CH-8093, Switzerland.

|December 17, 2020

Summary

This summary is machine-generated.

Researchers developed a new method to study how protein complexes assemble. They found that the nuclear pore complex (NPC) in yeast forms hierarchically, with subcomplexes assembling first and then maturing over an hour.

Keywords:

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

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Area of Science:

Cell Biology
Biochemistry
Structural Biology

Background:

Protein complexes are essential for cellular functions.
Characterizing the assembly dynamics of large protein complexes like the nuclear pore complex (NPC) remains challenging.
Understanding protein complex assembly is key to deciphering cellular processes.

Purpose of the Study:

To develop a high-throughput strategy for analyzing the native assembly kinetics of protein complexes.
To characterize the co-assembly dynamics of nucleoporins (NUPs) within the yeast NPC.
To reveal principles of NPC biogenesis and protein complex assembly.

Main Methods:

Developed a high-throughput strategy to analyze native assembly kinetics.
Applied the method to study the co-assembly of 320 nucleoporin pairs in yeast.
Investigated the timescales and hierarchical assembly of the nuclear pore complex.

Main Results:

Identified varying co-assembly rates among nucleoporins, from rapid exchange to lengthy maturation.
Revealed a hierarchical NPC biogenesis model: subcomplex formation (minutes) followed by center-to-periphery assembly (∼1 hour).
Discovered that Mlp1 (a nucleoporin) joins late, preferentially associating with mature NPCs.

Conclusions:

The developed high-throughput strategy enables analysis of protein complex assembly dynamics.
NPC assembly follows a hierarchical, time-dependent process.
The findings provide insights into the dynamic assembly of large cellular machinery and can be applied to other multiprotein assemblies.