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Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

Ciesielski SJ, Schilke B, Marszalek J, Craig EA - Mol. Biol. Cell (2016)

Bottom Line: Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu.Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1.Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.

No MeSH data available.


Related in: MedlinePlus

LON protease degrades Isu in vitro. Isu1 (7.5 μM) was incubated alone or with human LON WT or LON S_A (1.25 μM) in standard reaction conditions or in the absence of ATP and MgCl2 (–ATP/Mg2+). At indicated times, aliquots were removed, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom).
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Figure 2: LON protease degrades Isu in vitro. Isu1 (7.5 μM) was incubated alone or with human LON WT or LON S_A (1.25 μM) in standard reaction conditions or in the absence of ATP and MgCl2 (–ATP/Mg2+). At indicated times, aliquots were removed, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom).

Mentions: Encouraged by the ability of human LON protease to substitute for yeast Pim1 in vivo, we purified it for use in in vitro degradation assays. Having confirmed that Isu1 was stable upon incubation at 30°C (Figure 2), we mixed it with a substoichiometric concentration of LON protease (7.5 μM Isu1; 1.25 μM LON monomer) and removed aliquots over a 30-min time course. The amount of full-length Isu1 present in the reaction decreased with time. Only 4% remained after 30 min. To verify that the decrease was due to LON-dependent proteolysis, we performed additional control experiments (Figure 2). First, since Lon-type proteases require ATP and Mg2+ ions for their proteolytic activity (Suzuki et al., 1994; Van Dyck et al., 1994), we performed the experiment as described, except that we left out ATP and Mg2+ ions from the reaction buffer. No decrease in the amount of Isu1 occurred over the 30-min time course. Second, we purified and tested the human LON S_A variant. We observed no decrease in full-length Isu1. Taken together, these in vitro results establish that Isu1 is a substrate of the LON protease.


Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

Ciesielski SJ, Schilke B, Marszalek J, Craig EA - Mol. Biol. Cell (2016)

LON protease degrades Isu in vitro. Isu1 (7.5 μM) was incubated alone or with human LON WT or LON S_A (1.25 μM) in standard reaction conditions or in the absence of ATP and MgCl2 (–ATP/Mg2+). At indicated times, aliquots were removed, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom).
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Related In: Results  -  Collection

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Figure 2: LON protease degrades Isu in vitro. Isu1 (7.5 μM) was incubated alone or with human LON WT or LON S_A (1.25 μM) in standard reaction conditions or in the absence of ATP and MgCl2 (–ATP/Mg2+). At indicated times, aliquots were removed, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom).
Mentions: Encouraged by the ability of human LON protease to substitute for yeast Pim1 in vivo, we purified it for use in in vitro degradation assays. Having confirmed that Isu1 was stable upon incubation at 30°C (Figure 2), we mixed it with a substoichiometric concentration of LON protease (7.5 μM Isu1; 1.25 μM LON monomer) and removed aliquots over a 30-min time course. The amount of full-length Isu1 present in the reaction decreased with time. Only 4% remained after 30 min. To verify that the decrease was due to LON-dependent proteolysis, we performed additional control experiments (Figure 2). First, since Lon-type proteases require ATP and Mg2+ ions for their proteolytic activity (Suzuki et al., 1994; Van Dyck et al., 1994), we performed the experiment as described, except that we left out ATP and Mg2+ ions from the reaction buffer. No decrease in the amount of Isu1 occurred over the 30-min time course. Second, we purified and tested the human LON S_A variant. We observed no decrease in full-length Isu1. Taken together, these in vitro results establish that Isu1 is a substrate of the LON protease.

Bottom Line: Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu.Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1.Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.

No MeSH data available.


Related in: MedlinePlus