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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-type protease Pim1 regulates Isu levels in vivo and can be substituted for by human LON. (A) Equal amounts of whole-cell lysates, prepared from a WT strain and strains lacking mitochondrial protease Pim1 (pim1Δ), i-AAA (yme1Δ), m-AAA (yta12Δ), and Oma1 (oma1Δ), respectively, were separated by SDS–PAGE and subjected to immunoblot analysis with Isu-specific antibodies and, as a loading control, Mge1-specific antibodies. (B) WT or pim1Δ cells, transformed with plasmid vector (empty) or vector encoding yeast (PIM1) or human (LON) Lon-type protease or their proteolytically inactive variants (pim1 S_A or lon S_A, respectively) were plated as 1:10 serial dilutions on glucose-based synthetic medium (glucose) or glycerol-based rich medium (glycerol) and incubated at the indicated temperatures. (C) Equal amounts of whole-cell extracts prepared from strains described in B were separated by SDS–PAGE and probed with antibodies specific for Isu, Nfs1, and Jac1.
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Figure 1: Lon-type protease Pim1 regulates Isu levels in vivo and can be substituted for by human LON. (A) Equal amounts of whole-cell lysates, prepared from a WT strain and strains lacking mitochondrial protease Pim1 (pim1Δ), i-AAA (yme1Δ), m-AAA (yta12Δ), and Oma1 (oma1Δ), respectively, were separated by SDS–PAGE and subjected to immunoblot analysis with Isu-specific antibodies and, as a loading control, Mge1-specific antibodies. (B) WT or pim1Δ cells, transformed with plasmid vector (empty) or vector encoding yeast (PIM1) or human (LON) Lon-type protease or their proteolytically inactive variants (pim1 S_A or lon S_A, respectively) were plated as 1:10 serial dilutions on glucose-based synthetic medium (glucose) or glycerol-based rich medium (glycerol) and incubated at the indicated temperatures. (C) Equal amounts of whole-cell extracts prepared from strains described in B were separated by SDS–PAGE and probed with antibodies specific for Isu, Nfs1, and Jac1.

Mentions: Isu levels, in comparison to the wild-type (WT) strain, are markedly higher in yeast cells lacking the mitochondrial Lon-type protease Pim1 (Song et al., 2012), the only soluble protease in the matrix. We used immunoblot analysis to test whether proteases of the inner membrane known to have a range of substrate specificities might also be important for Isu degradation (Koppen and Langer, 2007). We measured Isu levels in yta12Δ and oma1Δ cells, which lack m-AAA and Oma1 proteolytic activity, respectively. As controls, we included a deletion of YME1, which encodes the i-AAA protease whose active site faces the intermembrane space, as well as a deletion of PIM1. Only the pim1Δ cells had elevated Isu levels compared with WT cells (Figure 1A), pointing to the Pim1 protease as the key regulator of Isu abundance in vivo.


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-type protease Pim1 regulates Isu levels in vivo and can be substituted for by human LON. (A) Equal amounts of whole-cell lysates, prepared from a WT strain and strains lacking mitochondrial protease Pim1 (pim1Δ), i-AAA (yme1Δ), m-AAA (yta12Δ), and Oma1 (oma1Δ), respectively, were separated by SDS–PAGE and subjected to immunoblot analysis with Isu-specific antibodies and, as a loading control, Mge1-specific antibodies. (B) WT or pim1Δ cells, transformed with plasmid vector (empty) or vector encoding yeast (PIM1) or human (LON) Lon-type protease or their proteolytically inactive variants (pim1 S_A or lon S_A, respectively) were plated as 1:10 serial dilutions on glucose-based synthetic medium (glucose) or glycerol-based rich medium (glycerol) and incubated at the indicated temperatures. (C) Equal amounts of whole-cell extracts prepared from strains described in B were separated by SDS–PAGE and probed with antibodies specific for Isu, Nfs1, and Jac1.
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Related In: Results  -  Collection

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Figure 1: Lon-type protease Pim1 regulates Isu levels in vivo and can be substituted for by human LON. (A) Equal amounts of whole-cell lysates, prepared from a WT strain and strains lacking mitochondrial protease Pim1 (pim1Δ), i-AAA (yme1Δ), m-AAA (yta12Δ), and Oma1 (oma1Δ), respectively, were separated by SDS–PAGE and subjected to immunoblot analysis with Isu-specific antibodies and, as a loading control, Mge1-specific antibodies. (B) WT or pim1Δ cells, transformed with plasmid vector (empty) or vector encoding yeast (PIM1) or human (LON) Lon-type protease or their proteolytically inactive variants (pim1 S_A or lon S_A, respectively) were plated as 1:10 serial dilutions on glucose-based synthetic medium (glucose) or glycerol-based rich medium (glycerol) and incubated at the indicated temperatures. (C) Equal amounts of whole-cell extracts prepared from strains described in B were separated by SDS–PAGE and probed with antibodies specific for Isu, Nfs1, and Jac1.
Mentions: Isu levels, in comparison to the wild-type (WT) strain, are markedly higher in yeast cells lacking the mitochondrial Lon-type protease Pim1 (Song et al., 2012), the only soluble protease in the matrix. We used immunoblot analysis to test whether proteases of the inner membrane known to have a range of substrate specificities might also be important for Isu degradation (Koppen and Langer, 2007). We measured Isu levels in yta12Δ and oma1Δ cells, which lack m-AAA and Oma1 proteolytic activity, respectively. As controls, we included a deletion of YME1, which encodes the i-AAA protease whose active site faces the intermembrane space, as well as a deletion of PIM1. Only the pim1Δ cells had elevated Isu levels compared with WT cells (Figure 1A), pointing to the Pim1 protease as the key regulator of Isu abundance in vivo.

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