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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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Model for regulation of Drosophila katanin by its non-catalytic domains.Schematic representation of the proposed contributions of the non-catalytic domains of Drosophila katanin to its proteasome-dependent degradation and microtubule association. Converging solid lines indicate cooperation between the MIT domain and linker region of Kat60 and solid lines with arrowheads indicate enhancement of the proteasome-dependent degradation and microtubule association of Kat60 by its MIT domain and linker region. The solid line with a blunt arrowhead indicates reduction of the proteasome-dependent degradation of Kat60 by Kat80 and the dashed line with an arrowhead indicates alteration of the microtubule association of Kat60 by Kat80. See text for details.
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pone.0123912.g008: Model for regulation of Drosophila katanin by its non-catalytic domains.Schematic representation of the proposed contributions of the non-catalytic domains of Drosophila katanin to its proteasome-dependent degradation and microtubule association. Converging solid lines indicate cooperation between the MIT domain and linker region of Kat60 and solid lines with arrowheads indicate enhancement of the proteasome-dependent degradation and microtubule association of Kat60 by its MIT domain and linker region. The solid line with a blunt arrowhead indicates reduction of the proteasome-dependent degradation of Kat60 by Kat80 and the dashed line with an arrowhead indicates alteration of the microtubule association of Kat60 by Kat80. See text for details.

Mentions: Collectively, our data suggest a model for how the non-catalytic domains of Drosophila katanin regulate microtubule severing (Fig 8). First, we speculate that the MIT domain of Kat60 physically interacts with Kat80—a well-established mode of interaction between katanin catalytic and regulatory subunits from several species [15,16]—and that the primary function of this interaction is to regulate the abundance of Kat60 itself. Our data and those of others [34,38,39] are consistent with a mechanism in which the MIT domain and linker region of Kat60 enhance its proteasome-dependent degradation by promoting interactions with ubiquitin ligase complexes, whereas Kat80 reduces the proteasome-dependent degradation of Kat60 by antagonizing such interactions. This mechanism could serve to maintain stoichiometric levels of Kat60 and Kat80 to ensure proper heterodimeric complex formation. In addition to regulating the abundance of Kat60, our data also demonstrate that the MIT domain and linker region of Kat60 regulate its microtubule-disassembly activity. In support of the katanin assembly model proposed by Hartman and Vale [17], our data suggest that the MIT domain and linker region of Kat60 enhance its initial targeting to microtubules. We speculate that the MIT domain and linker region of Kat60 also influence the subsequent balancing between Kat60-microtubule and Kat60-Kat60 interactions, however our data indicate that the MIT domain cannot be required for these interactions. On the other hand, our data suggest that Kat80 does not affect the microtubule-disassembly activity of Kat60 despite altering the association of Kat60 with microtubules. Previous studies of katanin from several species have shown that the katanin regulatory subunit potently stimulates the microtubule-severing activity of the katanin catalytic subunit in vitro [25] and that the heterodimeric complex exhibits distinct microtubule-binding properties in vitro [40]. Thus, we speculate Kat80 might function to target Kat60 to unique structural features or post-translational modifications of microtubules where it requires activation via some regulatory input to stimulate microtubule severing. Future work will focus on determining the functional importance of the microtubule-binding activity of Kat80 in living cells.


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

Grode KD, Rogers SL - PLoS ONE (2015)

Model for regulation of Drosophila katanin by its non-catalytic domains.Schematic representation of the proposed contributions of the non-catalytic domains of Drosophila katanin to its proteasome-dependent degradation and microtubule association. Converging solid lines indicate cooperation between the MIT domain and linker region of Kat60 and solid lines with arrowheads indicate enhancement of the proteasome-dependent degradation and microtubule association of Kat60 by its MIT domain and linker region. The solid line with a blunt arrowhead indicates reduction of the proteasome-dependent degradation of Kat60 by Kat80 and the dashed line with an arrowhead indicates alteration of the microtubule association of Kat60 by Kat80. See text for details.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123912.g008: Model for regulation of Drosophila katanin by its non-catalytic domains.Schematic representation of the proposed contributions of the non-catalytic domains of Drosophila katanin to its proteasome-dependent degradation and microtubule association. Converging solid lines indicate cooperation between the MIT domain and linker region of Kat60 and solid lines with arrowheads indicate enhancement of the proteasome-dependent degradation and microtubule association of Kat60 by its MIT domain and linker region. The solid line with a blunt arrowhead indicates reduction of the proteasome-dependent degradation of Kat60 by Kat80 and the dashed line with an arrowhead indicates alteration of the microtubule association of Kat60 by Kat80. See text for details.
Mentions: Collectively, our data suggest a model for how the non-catalytic domains of Drosophila katanin regulate microtubule severing (Fig 8). First, we speculate that the MIT domain of Kat60 physically interacts with Kat80—a well-established mode of interaction between katanin catalytic and regulatory subunits from several species [15,16]—and that the primary function of this interaction is to regulate the abundance of Kat60 itself. Our data and those of others [34,38,39] are consistent with a mechanism in which the MIT domain and linker region of Kat60 enhance its proteasome-dependent degradation by promoting interactions with ubiquitin ligase complexes, whereas Kat80 reduces the proteasome-dependent degradation of Kat60 by antagonizing such interactions. This mechanism could serve to maintain stoichiometric levels of Kat60 and Kat80 to ensure proper heterodimeric complex formation. In addition to regulating the abundance of Kat60, our data also demonstrate that the MIT domain and linker region of Kat60 regulate its microtubule-disassembly activity. In support of the katanin assembly model proposed by Hartman and Vale [17], our data suggest that the MIT domain and linker region of Kat60 enhance its initial targeting to microtubules. We speculate that the MIT domain and linker region of Kat60 also influence the subsequent balancing between Kat60-microtubule and Kat60-Kat60 interactions, however our data indicate that the MIT domain cannot be required for these interactions. On the other hand, our data suggest that Kat80 does not affect the microtubule-disassembly activity of Kat60 despite altering the association of Kat60 with microtubules. Previous studies of katanin from several species have shown that the katanin regulatory subunit potently stimulates the microtubule-severing activity of the katanin catalytic subunit in vitro [25] and that the heterodimeric complex exhibits distinct microtubule-binding properties in vitro [40]. Thus, we speculate Kat80 might function to target Kat60 to unique structural features or post-translational modifications of microtubules where it requires activation via some regulatory input to stimulate microtubule severing. Future work will focus on determining the functional importance of the microtubule-binding activity of Kat80 in living cells.

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