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Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy a
a
Amélie Bernard & Daniel J. Klionsky a
Life Sciences Institute, and the Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA Accepted author version posted online: 09 Mar 2015.
Click for updates To cite this article: Amélie Bernard & Daniel J. Klionsky (2015): Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy, Autophagy To link to this article: http://dx.doi.org/10.1080/15548627.2015.1018503
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy
ip t cr
us
Life Sciences Institute, and the Department of Molecular, Cellular and Developmental
an
Biology, University of Michigan, Ann Arbor, MI 48109, USA
ed
M
*Correspondence: [email protected]; Tel 734-615-6556; Fax 734-647-9702
ce
pt
Key words: autophagy, lysosome, stress, vacuole, yeast
To maintain proper cellular homeostasis, the magnitude of autophagy
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
Amélie Bernard and Daniel J. Klionsky*
activity has to be finely tuned in response to environmental changes. Many aspects of autophagy regulation have been extensively studied: pathways integrating signals through the master regulators TORC1 and PKA lead to multiple posttranslational modifications affecting the functions, protein-protein interactions and localization of
1
Atg proteins. The expression of several ATG genes increases sharply upon autophagy induction conditions, and defects in ATG gene expression are associated with various diseases, pointing to the importance of transcriptional regulation of autophagy. Yet, how changes in ATG gene expression affect the rate of autophagy is
ip t
not well characterized, and transcriptional regulators of the autophagy pathway
cr
several ATG genes in a library of DNA-binding protein mutants. This led to the
ce
pt
ed
M
an
us
identification of Rph1 as a master transcriptional regulator of autophagy.
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
remain largely unknown. To identify such regulators, we analyzed the expression of
2
Cells lacking RPH1 show a higher expression of several ATG genes and their corresponding proteins specifically in physiological conditions. This upregulation is associated with an increase in autophagy flux soon after nitrogen starvation. Conversely,
ip t
overexpressing RPH1 blocks the induction of expression normally observed for these
cr
Together these data demonstrated that Rph1 functions as a negative regulator of
us
autophagy by repressing the expression of ATG genes in rich conditions. The expression of ATG8 and ATG9 is highly increased upon autophagy induction, and the products of
an
these genes control the size and number of autophagosomes, respectively.
Here, we show that although RPH1 regulates several ATG genes including ATG8
M
and ATG9, it particularly affects the expression of ATG7. Lowering the level of Atg7
ed
leads to a reduced autophagy activity suggesting that a concomitant increase of Atg7 and Atg8 is required for proper autophagy induction upon nitrogen starvation. Consistent with
pt
this idea is the finding that the single overexpression of ATG7 is not sufficient to promote autophagy, suggesting that the later requires a concerted upregulation of multiple ATG
ce
genes. Our results further indicate that the overexpression of RPH1 causes a drastic
reduction in the number of autophagosomes, but does not affect their size. These results are surprising considering that a reduction in the level of Atg8 was previously associated
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
genes after starvation, and consequently results in a large defect in autophagy activity.
with a reduction in autophagosome size. However, we hypothesize that here, a block in the induction of multiple ATG genes, including ATG9, might slow the overall autophagosome biogenesis process thus allowing the formation of full-sized vesicles.
3
How does Rph1 control ATG gene expression? Rph1 is a DNA-binding protein, which contains a Jumonji C histone demethylase catalytic domain. Cells deprived of trimethylation at H3K36 or cells expressing a catalytically inactive mutant of Rph1 have normal autophagic capacity demonstrating that Rph1 functions mostly independently of
ip t
its histone demethylase activity in regulating autophagy. Conversely, an Rph1 mutant
cr
able to rescue the rph1∆ phenotype. These results suggest that Rph1 controls the
us
expression of ATG genes by directly binding to their promoters thus restricting the access of potential activators, or of the general transcriptional machinery.
an
How is Rph1 function released to promote autophagy under starvation conditions? We noticed that the RPH1 deletion strain shows higher expression of ATG genes only in
M
rich conditions suggesting that the repressive effect of Rph1 is released after nitrogen
ed
starvation. Furthermore the fact that overexpressing the protein blocked the induction of ATG gene expression upon nutrient limitation pointed to the strict requirement of such a
pt
release for the proper induction of autophagy. We found that Rph1 is highly phosphorylated upon nitrogen starvation and we identified some of the residues involved
ce
in the posttranslational modification; these data indicate that a reduction in Rph1 phosphorylation leads to a partial block in autophagy flux. In addition, we identified Rim15 as the kinase mediating Rph1 phosphorylation under these conditions, thereby
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
lacking its DNA-binding domain, which is not able to bind the ATG7 promoter, is not
inhibiting its function and allowing for autophagy induction. Rim15 controls autophagy, notably by integrating signals from TORC1 and PKA. TORC1 prevents Rim15 nuclear localization through its phosphorylation by Sch9, whereas PKA directly phosphorylates Rim15 thereby inhibiting its kinase activity. If the posttranslational regulation of
4
autophagy involving the master regulator TORC1 is well defined, its transcriptional effect on autophagy is less understood. Moreover, the mechanisms by which PKA and Sch9 inhibit autophagy are still mostly elusive; Rim15 positively regulates autophagy upon PKA-Sch9 inactivation, yet little was known about the downstream effectors in this
ip t
signaling pathway. Here, we characterize Rph1 as one of these effectors and uncover a
cr
Rim15-dependent phosphorylation of Ume6 after nitrogen starvation, which leads to a
us
release of its repression on the expression of ATG8 and an induction of autophagy. Finally, our work showed that the function of Rph1 is conserved in higher
an
eukaryotes. A reduction in the expression of one mammalian homolog of RPH1, KDM4A, leads to an increase of autophagy flux whereas, conversely, its overexpression
M
blocks autophagy activity. The level of KDM4A decreases after autophagy induction
ed
while the phosphorylation of the protein is increased suggesting that, like Rph1, the inhibition of KDM4A activity acts as a switch to promote autophagy in stress conditions.
pt
Rph1 was previously identified as a negative regulator of several DNA-damage inducible genes and is therefore though to be part of the signaling pathway leading to the
ce
cell response upon genotoxic stress. Rph1 is phosphorylated in these conditions as well as upon osmotic stress, suggesting that it might mediate broader stress signal responses. DNA damage induces autophagy in both yeast and mammalian cells; it would therefore
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
Rim15-dependent Rph1-mediated transcriptional control of autophagy similar to the
be of interest to determine whether the role of Rph1/KDM4A in autophagy induction is conserved in these conditions.
This work was supported by NIH grant GM053396 to DJK.
5
Autophagy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kaup20
Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy a
a
Amélie Bernard & Daniel J. Klionsky a
Life Sciences Institute, and the Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA Accepted author version posted online: 09 Mar 2015.
Click for updates To cite this article: Amélie Bernard & Daniel J. Klionsky (2015): Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy, Autophagy To link to this article: http://dx.doi.org/10.1080/15548627.2015.1018503
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy
ip t cr
us
Life Sciences Institute, and the Department of Molecular, Cellular and Developmental
an
Biology, University of Michigan, Ann Arbor, MI 48109, USA
ed
M
*Correspondence: [email protected]; Tel 734-615-6556; Fax 734-647-9702
ce
pt
Key words: autophagy, lysosome, stress, vacuole, yeast
To maintain proper cellular homeostasis, the magnitude of autophagy
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
Amélie Bernard and Daniel J. Klionsky*
activity has to be finely tuned in response to environmental changes. Many aspects of autophagy regulation have been extensively studied: pathways integrating signals through the master regulators TORC1 and PKA lead to multiple posttranslational modifications affecting the functions, protein-protein interactions and localization of
1
Atg proteins. The expression of several ATG genes increases sharply upon autophagy induction conditions, and defects in ATG gene expression are associated with various diseases, pointing to the importance of transcriptional regulation of autophagy. Yet, how changes in ATG gene expression affect the rate of autophagy is
ip t
not well characterized, and transcriptional regulators of the autophagy pathway
cr
several ATG genes in a library of DNA-binding protein mutants. This led to the
ce
pt
ed
M
an
us
identification of Rph1 as a master transcriptional regulator of autophagy.
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
remain largely unknown. To identify such regulators, we analyzed the expression of
2
Cells lacking RPH1 show a higher expression of several ATG genes and their corresponding proteins specifically in physiological conditions. This upregulation is associated with an increase in autophagy flux soon after nitrogen starvation. Conversely,
ip t
overexpressing RPH1 blocks the induction of expression normally observed for these
cr
Together these data demonstrated that Rph1 functions as a negative regulator of
us
autophagy by repressing the expression of ATG genes in rich conditions. The expression of ATG8 and ATG9 is highly increased upon autophagy induction, and the products of
an
these genes control the size and number of autophagosomes, respectively.
Here, we show that although RPH1 regulates several ATG genes including ATG8
M
and ATG9, it particularly affects the expression of ATG7. Lowering the level of Atg7
ed
leads to a reduced autophagy activity suggesting that a concomitant increase of Atg7 and Atg8 is required for proper autophagy induction upon nitrogen starvation. Consistent with
pt
this idea is the finding that the single overexpression of ATG7 is not sufficient to promote autophagy, suggesting that the later requires a concerted upregulation of multiple ATG
ce
genes. Our results further indicate that the overexpression of RPH1 causes a drastic
reduction in the number of autophagosomes, but does not affect their size. These results are surprising considering that a reduction in the level of Atg8 was previously associated
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
genes after starvation, and consequently results in a large defect in autophagy activity.
with a reduction in autophagosome size. However, we hypothesize that here, a block in the induction of multiple ATG genes, including ATG9, might slow the overall autophagosome biogenesis process thus allowing the formation of full-sized vesicles.
3
How does Rph1 control ATG gene expression? Rph1 is a DNA-binding protein, which contains a Jumonji C histone demethylase catalytic domain. Cells deprived of trimethylation at H3K36 or cells expressing a catalytically inactive mutant of Rph1 have normal autophagic capacity demonstrating that Rph1 functions mostly independently of
ip t
its histone demethylase activity in regulating autophagy. Conversely, an Rph1 mutant
cr
able to rescue the rph1∆ phenotype. These results suggest that Rph1 controls the
us
expression of ATG genes by directly binding to their promoters thus restricting the access of potential activators, or of the general transcriptional machinery.
an
How is Rph1 function released to promote autophagy under starvation conditions? We noticed that the RPH1 deletion strain shows higher expression of ATG genes only in
M
rich conditions suggesting that the repressive effect of Rph1 is released after nitrogen
ed
starvation. Furthermore the fact that overexpressing the protein blocked the induction of ATG gene expression upon nutrient limitation pointed to the strict requirement of such a
pt
release for the proper induction of autophagy. We found that Rph1 is highly phosphorylated upon nitrogen starvation and we identified some of the residues involved
ce
in the posttranslational modification; these data indicate that a reduction in Rph1 phosphorylation leads to a partial block in autophagy flux. In addition, we identified Rim15 as the kinase mediating Rph1 phosphorylation under these conditions, thereby
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
lacking its DNA-binding domain, which is not able to bind the ATG7 promoter, is not
inhibiting its function and allowing for autophagy induction. Rim15 controls autophagy, notably by integrating signals from TORC1 and PKA. TORC1 prevents Rim15 nuclear localization through its phosphorylation by Sch9, whereas PKA directly phosphorylates Rim15 thereby inhibiting its kinase activity. If the posttranslational regulation of
4
autophagy involving the master regulator TORC1 is well defined, its transcriptional effect on autophagy is less understood. Moreover, the mechanisms by which PKA and Sch9 inhibit autophagy are still mostly elusive; Rim15 positively regulates autophagy upon PKA-Sch9 inactivation, yet little was known about the downstream effectors in this
ip t
signaling pathway. Here, we characterize Rph1 as one of these effectors and uncover a
cr
Rim15-dependent phosphorylation of Ume6 after nitrogen starvation, which leads to a
us
release of its repression on the expression of ATG8 and an induction of autophagy. Finally, our work showed that the function of Rph1 is conserved in higher
an
eukaryotes. A reduction in the expression of one mammalian homolog of RPH1, KDM4A, leads to an increase of autophagy flux whereas, conversely, its overexpression
M
blocks autophagy activity. The level of KDM4A decreases after autophagy induction
ed
while the phosphorylation of the protein is increased suggesting that, like Rph1, the inhibition of KDM4A activity acts as a switch to promote autophagy in stress conditions.
pt
Rph1 was previously identified as a negative regulator of several DNA-damage inducible genes and is therefore though to be part of the signaling pathway leading to the
ce
cell response upon genotoxic stress. Rph1 is phosphorylated in these conditions as well as upon osmotic stress, suggesting that it might mediate broader stress signal responses. DNA damage induces autophagy in both yeast and mammalian cells; it would therefore
Ac
Downloaded by [University of Sussex Library] at 15:51 26 March 2015
Rim15-dependent Rph1-mediated transcriptional control of autophagy similar to the
be of interest to determine whether the role of Rph1/KDM4A in autophagy induction is conserved in these conditions.
This work was supported by NIH grant GM053396 to DJK.
5
