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The targeted destruction of specific proteins is an important means of regulation for many cellular pathways. This is typically managed through the process called ubiquitination, in which doomed proteins are chemically marked for entry into a degradation pathway by protein complexes known as ubiquitin ligases.

“Although the identification of substrates is essential for our understanding of cellular regulatory mechanisms involving ubiquitination, identifying them is quite difficult,” explains Tsutomu Kishi of the Advanced Science Institute in Wako, whose work on ubiquitin ligase target recognition via subunits known as ‘F-box proteins’ has been impeded by the limitations of existing tools for protein–protein interaction analysis.

One popular method is the ‘two-hybrid’ system, which uses a gene-activating protein that has been split into two pieces: one capable of binding to a target DNA sequence, and one capable of inducing activation. The first piece is fused to a ‘bait’ protein, while the second piece is fused to various ‘prey’ proteins; both bait and prey are then introduced into yeast cells with an indicator gene containing an appropriate binding site for the bait. The indicator is only turned on if the DNA-binding domain and gene activation domain become linked via prey–bait interaction, making it easy to identify such associations.

When working with ubiquitination targets, however, prey fusions are in danger of being marked for rapid destruction by the host cell before interactions can be detected. Kishi and colleagues therefore modified the assay so that it could be performed under conditions in which the relevant degradation pathways are disabled, enabling straightforward two-hybrid analysis of substrates from these pathways1.

Kishi’s team applied their method to Cdc4, a component of the SCFCdc4 ubiquitin ligase complex. They identified four interacting partners, but focused on Swi5, a protein that stimulates production of SIC1, a regulator that inhibits onset of S phase—and also a ubiquitination target. Subsequent experiments revealed that SCFCdc4 mediates a two-pronged process of SIC1 downregulation by first reducing levels of the activator protein Swi5, and then by inducing direct degradation of SIC1 itself.

These findings offer valuable insights into the regulation of the cell cycle and illustrate an important ‘indirect’ mechanism for ubiquitination-based regulation of protein levels via the targeting of relevant gene activators for destruction. They also demonstrate the effectiveness of a strategy that could be generalized for identifying other ubiquitination targets. “This methodology is widely applicable,” says Kishi, “and in collaboration with other groups, we have succeeded in identifying targets of other F-box proteins.”

Reference

1. Kishi, T., Ikeda, A., Koyama, N., Fukada, J. & Nagao, R. A refined two-hybrid system reveals that SCFCdc4-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry. Proceedings of the National Academy of Sciences USA 105, 14497–14502 (2008).

Saeko Okada | Quelle: ResearchSEA
Weitere Informationen: www.rikenresearch.riken.jp/research/628/

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