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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

Jac1 binding protects Isu from degradation in vitro. (A) Surface representation of Isu1 with residues involved in interaction with Nfs1 and Jac1 highlighted, based on previously published results (Majewska, Ciesielski, et al., 2013); prepared using PyMOL (www.schrodinger.com/pymol/). (B) Isu1 (7.5 μM) alone or after preincubation with 37.5 μM Jac1 WT or Jac1 LLY_AAA (Jac1 LLY) 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. (C) Reactions were performed as in B with increasing concentrations of Jac1 WT or Jac1 LLY_AAA (Jac1 LLY), with equimolar Jac1 to Isu1 concentration indicated as 1×. After 15 min, aliquots were collected, separated by SDS–PAGE, and stained. Amounts of full-length Isu1 were quantitated by densitometry and presented as a bar graph with the time-zero value set at 1. (D) Reactions were performed as in B but with Isu1 preloaded with zinc ions (Isu1(Zn)) instead of apo-Isu1. Amounts of full-length Isu1 at the indicated times were visualized, quantitated, and plotted as in B.
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Figure 4: Jac1 binding protects Isu from degradation in vitro. (A) Surface representation of Isu1 with residues involved in interaction with Nfs1 and Jac1 highlighted, based on previously published results (Majewska, Ciesielski, et al., 2013); prepared using PyMOL (www.schrodinger.com/pymol/). (B) Isu1 (7.5 μM) alone or after preincubation with 37.5 μM Jac1 WT or Jac1 LLY_AAA (Jac1 LLY) 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. (C) Reactions were performed as in B with increasing concentrations of Jac1 WT or Jac1 LLY_AAA (Jac1 LLY), with equimolar Jac1 to Isu1 concentration indicated as 1×. After 15 min, aliquots were collected, separated by SDS–PAGE, and stained. Amounts of full-length Isu1 were quantitated by densitometry and presented as a bar graph with the time-zero value set at 1. (D) Reactions were performed as in B but with Isu1 preloaded with zinc ions (Isu1(Zn)) instead of apo-Isu1. Amounts of full-length Isu1 at the indicated times were visualized, quantitated, and plotted as in B.

Mentions: The binding site of Nfs1 on Isu partially overlaps with that of Jac1 (Figure 4A; Majewska, Ciesielski, et al., 2013), raising the question of whether Jac1 binding can also protect Isu from degradation. To address this question, we preincubated Jac1 WT with Isu1 before adding LON protease. The presence of Jac1 WT resulted in significant inhibition of Isu1 degradation (Figure 4B). Fifty-three percent of full-length Isu1 remained 10 min after LON addition when it was preincubated with a fivefold molar excess of Jac1, compared with 13% remaining in the absence of Jac1 (Figure 4B). To determine whether this protection of Isu1 from degradation depends on Jac1 binding to Isu1, analogous to our Nfs1 analysis, we took advantage of our previously isolated Jac1 variant that is defective in interaction with Isu1. Jac1 LLY_AAA does not form a stable complex with Isu1 due to alanine substitutions of residues Leu-105, Leu-109, and Tyr-163 (Ciesielski et al., 2012). Jac1 LLY_AAA did not significantly protect Isu1 from proteolysis. After 30 min, only 6% of Isu1 remained, comparable to the 4% remaining in the reaction without Jac1 (Figure 4B). Focusing on the 15-min time point, we assessed the concentration dependence of Jac1 protection. Only 8% more Isu1 remained when a sixfold excess of Jac1 LLY_AAA was used compared with that remaining in the reaction lacking Jac1, whereas in the presence of Jac1 WT, 45% of Isu1 remained (Figure 4C).


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)

Jac1 binding protects Isu from degradation in vitro. (A) Surface representation of Isu1 with residues involved in interaction with Nfs1 and Jac1 highlighted, based on previously published results (Majewska, Ciesielski, et al., 2013); prepared using PyMOL (www.schrodinger.com/pymol/). (B) Isu1 (7.5 μM) alone or after preincubation with 37.5 μM Jac1 WT or Jac1 LLY_AAA (Jac1 LLY) 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. (C) Reactions were performed as in B with increasing concentrations of Jac1 WT or Jac1 LLY_AAA (Jac1 LLY), with equimolar Jac1 to Isu1 concentration indicated as 1×. After 15 min, aliquots were collected, separated by SDS–PAGE, and stained. Amounts of full-length Isu1 were quantitated by densitometry and presented as a bar graph with the time-zero value set at 1. (D) Reactions were performed as in B but with Isu1 preloaded with zinc ions (Isu1(Zn)) instead of apo-Isu1. Amounts of full-length Isu1 at the indicated times were visualized, quantitated, and plotted as in B.
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Figure 4: Jac1 binding protects Isu from degradation in vitro. (A) Surface representation of Isu1 with residues involved in interaction with Nfs1 and Jac1 highlighted, based on previously published results (Majewska, Ciesielski, et al., 2013); prepared using PyMOL (www.schrodinger.com/pymol/). (B) Isu1 (7.5 μM) alone or after preincubation with 37.5 μM Jac1 WT or Jac1 LLY_AAA (Jac1 LLY) 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. (C) Reactions were performed as in B with increasing concentrations of Jac1 WT or Jac1 LLY_AAA (Jac1 LLY), with equimolar Jac1 to Isu1 concentration indicated as 1×. After 15 min, aliquots were collected, separated by SDS–PAGE, and stained. Amounts of full-length Isu1 were quantitated by densitometry and presented as a bar graph with the time-zero value set at 1. (D) Reactions were performed as in B but with Isu1 preloaded with zinc ions (Isu1(Zn)) instead of apo-Isu1. Amounts of full-length Isu1 at the indicated times were visualized, quantitated, and plotted as in B.
Mentions: The binding site of Nfs1 on Isu partially overlaps with that of Jac1 (Figure 4A; Majewska, Ciesielski, et al., 2013), raising the question of whether Jac1 binding can also protect Isu from degradation. To address this question, we preincubated Jac1 WT with Isu1 before adding LON protease. The presence of Jac1 WT resulted in significant inhibition of Isu1 degradation (Figure 4B). Fifty-three percent of full-length Isu1 remained 10 min after LON addition when it was preincubated with a fivefold molar excess of Jac1, compared with 13% remaining in the absence of Jac1 (Figure 4B). To determine whether this protection of Isu1 from degradation depends on Jac1 binding to Isu1, analogous to our Nfs1 analysis, we took advantage of our previously isolated Jac1 variant that is defective in interaction with Isu1. Jac1 LLY_AAA does not form a stable complex with Isu1 due to alanine substitutions of residues Leu-105, Leu-109, and Tyr-163 (Ciesielski et al., 2012). Jac1 LLY_AAA did not significantly protect Isu1 from proteolysis. After 30 min, only 6% of Isu1 remained, comparable to the 4% remaining in the reaction without Jac1 (Figure 4B). Focusing on the 15-min time point, we assessed the concentration dependence of Jac1 protection. Only 8% more Isu1 remained when a sixfold excess of Jac1 LLY_AAA was used compared with that remaining in the reaction lacking Jac1, whereas in the presence of Jac1 WT, 45% of Isu1 remained (Figure 4C).

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