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The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

Grode KD, Rogers SL - PLoS ONE (2015)

Bottom Line: First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation.The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60.Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

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Depletion of Kat80 reduces steady-state Kat60 levels in cells.(A and B) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP alone (A) or GFP and copper-inducible FLAG-Kat80 (B) that were treated with control (lane 1), Kat60 CDS (lane 2), or Kat80 CDS dsRNA (lane 3) for 7 days total. The cells described in B were also treated with 0.1 mM CuSO4 for 20 hours. Molecular weights (in Kd) are shown on the left.
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pone.0123912.g002: Depletion of Kat80 reduces steady-state Kat60 levels in cells.(A and B) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP alone (A) or GFP and copper-inducible FLAG-Kat80 (B) that were treated with control (lane 1), Kat60 CDS (lane 2), or Kat80 CDS dsRNA (lane 3) for 7 days total. The cells described in B were also treated with 0.1 mM CuSO4 for 20 hours. Molecular weights (in Kd) are shown on the left.

Mentions: To minimize the potential contribution of endogenous Kat60 and Kat80 to the single-cell measurements collected in each experiment, we devised an RNA interference (RNAi) strategy to specifically deplete the endogenous proteins from cells using double-stranded RNAs (dsRNA) that target the untranslated regions (UTR) of Kat60 and Kat80. Before evaluating the efficacy of this strategy, we first sought to determine if dsRNAs targeting the coding sequences (CDS) of Kat60 and Kat80 are effective in eliminating both proteins from cells. As an initial step, we treated cells stably expressing GFP alone with Kat60 or Kat80 CDS dsRNA and analyzed the steady-state levels of Kat60 by immunoblotting. Endogenous Kat60 was undetectable in cells treated with Kat60 CDS dsRNA and the levels of Kat60 in cells treated with Kat80 CDS dsRNA were dramatically reduced compared to those in cells treated with control dsRNA (Fig 2A), suggesting that Kat80 functions to stabilize Kat60. To verify the effectiveness of Kat80 CDS dsRNA treatment, we treated cells stably expressing GFP and copper-inducible FLAG-tagged Kat80 (FLAG-Kat80) with Kat60 or Kat80 CDS dsRNA prior to induction and analyzed the levels of FLAG-Kat80 by immunoblotting. FLAG-Kat80 was undetectable in cells treated with Kat80 CDS dsRNA and the levels of FLAG-Kat80 in cells treated with Kat60 CDS dsRNA were indistinguishable from those in cells treated with control dsRNA (Fig 2B). Thus, in addition to demonstrating that Kat60 and Kat80 CDS dsRNA treatments are effective in eliminating both proteins from cells, these findings provide strong evidence that Kat80 is required to stabilize Kat60 in cells. To our knowledge, the loss of Kat60 upon depletion of Kat80 represents the first example of instability of one katanin subunit in the absence of the other subunit.


The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

Grode KD, Rogers SL - PLoS ONE (2015)

Depletion of Kat80 reduces steady-state Kat60 levels in cells.(A and B) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP alone (A) or GFP and copper-inducible FLAG-Kat80 (B) that were treated with control (lane 1), Kat60 CDS (lane 2), or Kat80 CDS dsRNA (lane 3) for 7 days total. The cells described in B were also treated with 0.1 mM CuSO4 for 20 hours. Molecular weights (in Kd) are shown on the left.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401518&req=5

pone.0123912.g002: Depletion of Kat80 reduces steady-state Kat60 levels in cells.(A and B) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP alone (A) or GFP and copper-inducible FLAG-Kat80 (B) that were treated with control (lane 1), Kat60 CDS (lane 2), or Kat80 CDS dsRNA (lane 3) for 7 days total. The cells described in B were also treated with 0.1 mM CuSO4 for 20 hours. Molecular weights (in Kd) are shown on the left.
Mentions: To minimize the potential contribution of endogenous Kat60 and Kat80 to the single-cell measurements collected in each experiment, we devised an RNA interference (RNAi) strategy to specifically deplete the endogenous proteins from cells using double-stranded RNAs (dsRNA) that target the untranslated regions (UTR) of Kat60 and Kat80. Before evaluating the efficacy of this strategy, we first sought to determine if dsRNAs targeting the coding sequences (CDS) of Kat60 and Kat80 are effective in eliminating both proteins from cells. As an initial step, we treated cells stably expressing GFP alone with Kat60 or Kat80 CDS dsRNA and analyzed the steady-state levels of Kat60 by immunoblotting. Endogenous Kat60 was undetectable in cells treated with Kat60 CDS dsRNA and the levels of Kat60 in cells treated with Kat80 CDS dsRNA were dramatically reduced compared to those in cells treated with control dsRNA (Fig 2A), suggesting that Kat80 functions to stabilize Kat60. To verify the effectiveness of Kat80 CDS dsRNA treatment, we treated cells stably expressing GFP and copper-inducible FLAG-tagged Kat80 (FLAG-Kat80) with Kat60 or Kat80 CDS dsRNA prior to induction and analyzed the levels of FLAG-Kat80 by immunoblotting. FLAG-Kat80 was undetectable in cells treated with Kat80 CDS dsRNA and the levels of FLAG-Kat80 in cells treated with Kat60 CDS dsRNA were indistinguishable from those in cells treated with control dsRNA (Fig 2B). Thus, in addition to demonstrating that Kat60 and Kat80 CDS dsRNA treatments are effective in eliminating both proteins from cells, these findings provide strong evidence that Kat80 is required to stabilize Kat60 in cells. To our knowledge, the loss of Kat60 upon depletion of Kat80 represents the first example of instability of one katanin subunit in the absence of the other subunit.

Bottom Line: First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation.The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60.Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

ABSTRACT
Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

Show MeSH