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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

Nfs1 binding protects Isu from degradation in vitro. (A) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT or Nfs1 LM_AA (Nfs1 LM) was mixed with 1.25 μM human LON protease. After protease addition (i.e., time zero), aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from three independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD; in most cases, they are obscured by symbols. (B) Reactions were performed as in A with increasing concentrations of Nfs1 WT or Nfs1 LM_AA (Nfs1 LM), with equimolar Nfs1 and Isu1 concentration indicated as 1×. After 20 min, samples were collected, separated by SDS–PAGE, and stained. Isu1 amounts were quantitated by densitometry and presented as a bar graph with time-zero value set at 1. (C) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT (Nfs1), Yfh1, or both protein partners (Nfs1, Yfh1) was mixed with LON (1.25 μM). Aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD.
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Figure 3: Nfs1 binding protects Isu from degradation in vitro. (A) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT or Nfs1 LM_AA (Nfs1 LM) was mixed with 1.25 μM human LON protease. After protease addition (i.e., time zero), aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from three independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD; in most cases, they are obscured by symbols. (B) Reactions were performed as in A with increasing concentrations of Nfs1 WT or Nfs1 LM_AA (Nfs1 LM), with equimolar Nfs1 and Isu1 concentration indicated as 1×. After 20 min, samples were collected, separated by SDS–PAGE, and stained. Isu1 amounts were quantitated by densitometry and presented as a bar graph with time-zero value set at 1. (C) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT (Nfs1), Yfh1, or both protein partners (Nfs1, Yfh1) was mixed with LON (1.25 μM). Aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD.

Mentions: Because previously published in vivo results suggested that Nfs1 protects Isu from degradation (Andrew et al., 2008; Song et al., 2012), we decided to test the effect of Nfs1 addition on Isu1 degradation in vitro. Nfs1 was purified in complex with Isd11, a protein with which Nfs1 normally forms a heterodimer. Interaction with Isd11 is necessary to maintain Nfs1 in an active conformation (Adam et al., 2006). For simplicity, we refer to the Nfs1:Isd11 complex as Nfs1 throughout. We preincubated Isu1 with a threefold molar excess of Nfs1 before addition of LON to the reaction. Isu1 degradation was inhibited in the presence of Nfs1 (Figure 3A); after 10 min, 92% of Isu1 was intact, compared with 14% in the reaction without Nfs1. To determine whether stabilization of Isu1 depends on direct physical interaction with Nfs1, we took advantage of a previously isolated variant of Nfs1 having a reduced ability to bind Isu1 due to alanine substitutions of residues Leu-479 and Met-482 (Nfs1 LM_AA; Majewska, Ciesielski, et al., 2013). Nfs1 LM_AA did not protect Isu1 from degradation as effectively as Nfs1 WT. After 30 min, only 21% of Isu1 remained in the reaction with Nfs1 LM_AA, in contrast to 67% with Nfs1 WT (Figure 3A).


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)

Nfs1 binding protects Isu from degradation in vitro. (A) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT or Nfs1 LM_AA (Nfs1 LM) was mixed with 1.25 μM human LON protease. After protease addition (i.e., time zero), aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from three independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD; in most cases, they are obscured by symbols. (B) Reactions were performed as in A with increasing concentrations of Nfs1 WT or Nfs1 LM_AA (Nfs1 LM), with equimolar Nfs1 and Isu1 concentration indicated as 1×. After 20 min, samples were collected, separated by SDS–PAGE, and stained. Isu1 amounts were quantitated by densitometry and presented as a bar graph with time-zero value set at 1. (C) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT (Nfs1), Yfh1, or both protein partners (Nfs1, Yfh1) was mixed with LON (1.25 μM). Aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD.
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Figure 3: Nfs1 binding protects Isu from degradation in vitro. (A) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT or Nfs1 LM_AA (Nfs1 LM) was mixed with 1.25 μM human LON protease. After protease addition (i.e., time zero), aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from three independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD; in most cases, they are obscured by symbols. (B) Reactions were performed as in A with increasing concentrations of Nfs1 WT or Nfs1 LM_AA (Nfs1 LM), with equimolar Nfs1 and Isu1 concentration indicated as 1×. After 20 min, samples were collected, separated by SDS–PAGE, and stained. Isu1 amounts were quantitated by densitometry and presented as a bar graph with time-zero value set at 1. (C) Isu1 (7.5 μM) alone or after preincubation with 22.5 μM Nfs1 WT (Nfs1), Yfh1, or both protein partners (Nfs1, Yfh1) was mixed with LON (1.25 μM). Aliquots were collected at indicated times, separated by SDS–PAGE, and stained (top). Amounts of full-length Isu1 from independent experiments were quantitated by densitometry and plotted as relative units with the time-zero value set at 1 (bottom). Error bars are shown as ±SD.
Mentions: Because previously published in vivo results suggested that Nfs1 protects Isu from degradation (Andrew et al., 2008; Song et al., 2012), we decided to test the effect of Nfs1 addition on Isu1 degradation in vitro. Nfs1 was purified in complex with Isd11, a protein with which Nfs1 normally forms a heterodimer. Interaction with Isd11 is necessary to maintain Nfs1 in an active conformation (Adam et al., 2006). For simplicity, we refer to the Nfs1:Isd11 complex as Nfs1 throughout. We preincubated Isu1 with a threefold molar excess of Nfs1 before addition of LON to the reaction. Isu1 degradation was inhibited in the presence of Nfs1 (Figure 3A); after 10 min, 92% of Isu1 was intact, compared with 14% in the reaction without Nfs1. To determine whether stabilization of Isu1 depends on direct physical interaction with Nfs1, we took advantage of a previously isolated variant of Nfs1 having a reduced ability to bind Isu1 due to alanine substitutions of residues Leu-479 and Met-482 (Nfs1 LM_AA; Majewska, Ciesielski, et al., 2013). Nfs1 LM_AA did not protect Isu1 from degradation as effectively as Nfs1 WT. After 30 min, only 21% of Isu1 remained in the reaction with Nfs1 LM_AA, in contrast to 67% with Nfs1 WT (Figure 3A).

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