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Snf1/AMP-activated protein kinase activates Arf3p to promote invasive yeast growth via a non-canonical GEF domain.

Hsu JW, Chen KJ, Lee FJ - Nat Commun (2015)

Bottom Line: Arf activation is accelerated by guanine nucleotide-exchange factors (GEFs) using the critical catalytic glutamate in all known Sec7 domain sequences.SNF1 is the yeast homologue of AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis.Thus, our study reveals a novel mechanism for regulating cellular responses to energy deprivation, in particular invasive cell growth, through direct Arf activation by Snf1/AMPK.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

ABSTRACT
Active GTP-bound Arf GTPases promote eukaryotic cell membrane trafficking and cytoskeletal remodelling. Arf activation is accelerated by guanine nucleotide-exchange factors (GEFs) using the critical catalytic glutamate in all known Sec7 domain sequences. Yeast Arf3p, a homologue of mammalian Arf6, is required for yeast invasive responses to glucose depletion. Here we identify Snf1p as a GEF that activates Arf3p when energy is limited. SNF1 is the yeast homologue of AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis. As activation of Arf3p does not depend on the Snf1p kinase domain, assay of regulatory domain fragments yield evidence that the C-terminal hydrophobic α-helix core of Snf1p is a non-canonical GEF for Arf3p activation. Thus, our study reveals a novel mechanism for regulating cellular responses to energy deprivation, in particular invasive cell growth, through direct Arf activation by Snf1/AMPK.

No MeSH data available.


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C-terminal regulatory domain of Snf1p is responsible for activating Arf3p in response to glucose depletion.(a) ARF3-GFP expressed under the control of the ADH1 promoter (CEN plasmid) was transformed into the indicated yeast cells. Transformants were grown to the exponential phase and inspected via microscopy. Scale bar, 5 μm. (b) The localization of Arf3p-GFP was observed in the indicated yeast cells grown in YPD or YP medium for 2 h. Scale bar, 5 μm. The fluorescence intensity (F.I.) profiles from the line scan are shown in each lower panel. The plasma membrane association ratio of Arf3p was quantified using Axio Vision Rel. 4.2 software. The F.I.'s of the plasma membrane signals were summed and divided by the whole-cell signals (n=50) as described in Methods section. Data are reported as the mean±s.d. **P<0.01; Student's t-test. (c) Active forms of Arf3p were precipitated by GST-Afi1N in the indicated cells and immunoblotted for Arf3p. Quantitative analysis of active Arf3p is presented in the right panel. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. Vec, vector; WT, wild type.
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f4: C-terminal regulatory domain of Snf1p is responsible for activating Arf3p in response to glucose depletion.(a) ARF3-GFP expressed under the control of the ADH1 promoter (CEN plasmid) was transformed into the indicated yeast cells. Transformants were grown to the exponential phase and inspected via microscopy. Scale bar, 5 μm. (b) The localization of Arf3p-GFP was observed in the indicated yeast cells grown in YPD or YP medium for 2 h. Scale bar, 5 μm. The fluorescence intensity (F.I.) profiles from the line scan are shown in each lower panel. The plasma membrane association ratio of Arf3p was quantified using Axio Vision Rel. 4.2 software. The F.I.'s of the plasma membrane signals were summed and divided by the whole-cell signals (n=50) as described in Methods section. Data are reported as the mean±s.d. **P<0.01; Student's t-test. (c) Active forms of Arf3p were precipitated by GST-Afi1N in the indicated cells and immunoblotted for Arf3p. Quantitative analysis of active Arf3p is presented in the right panel. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. Vec, vector; WT, wild type.

Mentions: We next assessed the relative roles of the two Arf3p GEFs, Snf1p and Yel1p. The activation of Arf3p by Snf1p does not require the presence of Yel1p (Fig. 1c). Moreover, SNF1-C overexpression in yel1Δ cells increased the targeting of Arf3p to the plasma membrane, which is a signature effect of Arf3p activation in yeast1213 (Fig. 4a). We also assessed the activation status of Arf3p in the different deletion strains by examining the distribution of Arf3p on the plasma membrane and the level of active Arf3p using effector pull-down assays (Fig. 4b,c). We found that snf1Δ cells could not increase Arf3p activation on glucose deprivation, whereas yel1Δ cells had this ability. Moreover, the level of active Arf3p was reduced in yel1snf1Δ cells compared with that in the single-deletion mutants. Thus, the results suggest that the two Arf3GEFs function independently in controlling Arf3p activation; both Snf1p and Yel1p activate Arf3p during glucose-starvation conditions, whereas only Yel1p activates Arf3p during vegetative growth.


Snf1/AMP-activated protein kinase activates Arf3p to promote invasive yeast growth via a non-canonical GEF domain.

Hsu JW, Chen KJ, Lee FJ - Nat Commun (2015)

C-terminal regulatory domain of Snf1p is responsible for activating Arf3p in response to glucose depletion.(a) ARF3-GFP expressed under the control of the ADH1 promoter (CEN plasmid) was transformed into the indicated yeast cells. Transformants were grown to the exponential phase and inspected via microscopy. Scale bar, 5 μm. (b) The localization of Arf3p-GFP was observed in the indicated yeast cells grown in YPD or YP medium for 2 h. Scale bar, 5 μm. The fluorescence intensity (F.I.) profiles from the line scan are shown in each lower panel. The plasma membrane association ratio of Arf3p was quantified using Axio Vision Rel. 4.2 software. The F.I.'s of the plasma membrane signals were summed and divided by the whole-cell signals (n=50) as described in Methods section. Data are reported as the mean±s.d. **P<0.01; Student's t-test. (c) Active forms of Arf3p were precipitated by GST-Afi1N in the indicated cells and immunoblotted for Arf3p. Quantitative analysis of active Arf3p is presented in the right panel. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. Vec, vector; WT, wild type.
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f4: C-terminal regulatory domain of Snf1p is responsible for activating Arf3p in response to glucose depletion.(a) ARF3-GFP expressed under the control of the ADH1 promoter (CEN plasmid) was transformed into the indicated yeast cells. Transformants were grown to the exponential phase and inspected via microscopy. Scale bar, 5 μm. (b) The localization of Arf3p-GFP was observed in the indicated yeast cells grown in YPD or YP medium for 2 h. Scale bar, 5 μm. The fluorescence intensity (F.I.) profiles from the line scan are shown in each lower panel. The plasma membrane association ratio of Arf3p was quantified using Axio Vision Rel. 4.2 software. The F.I.'s of the plasma membrane signals were summed and divided by the whole-cell signals (n=50) as described in Methods section. Data are reported as the mean±s.d. **P<0.01; Student's t-test. (c) Active forms of Arf3p were precipitated by GST-Afi1N in the indicated cells and immunoblotted for Arf3p. Quantitative analysis of active Arf3p is presented in the right panel. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. Vec, vector; WT, wild type.
Mentions: We next assessed the relative roles of the two Arf3p GEFs, Snf1p and Yel1p. The activation of Arf3p by Snf1p does not require the presence of Yel1p (Fig. 1c). Moreover, SNF1-C overexpression in yel1Δ cells increased the targeting of Arf3p to the plasma membrane, which is a signature effect of Arf3p activation in yeast1213 (Fig. 4a). We also assessed the activation status of Arf3p in the different deletion strains by examining the distribution of Arf3p on the plasma membrane and the level of active Arf3p using effector pull-down assays (Fig. 4b,c). We found that snf1Δ cells could not increase Arf3p activation on glucose deprivation, whereas yel1Δ cells had this ability. Moreover, the level of active Arf3p was reduced in yel1snf1Δ cells compared with that in the single-deletion mutants. Thus, the results suggest that the two Arf3GEFs function independently in controlling Arf3p activation; both Snf1p and Yel1p activate Arf3p during glucose-starvation conditions, whereas only Yel1p activates Arf3p during vegetative growth.

Bottom Line: Arf activation is accelerated by guanine nucleotide-exchange factors (GEFs) using the critical catalytic glutamate in all known Sec7 domain sequences.SNF1 is the yeast homologue of AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis.Thus, our study reveals a novel mechanism for regulating cellular responses to energy deprivation, in particular invasive cell growth, through direct Arf activation by Snf1/AMPK.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

ABSTRACT
Active GTP-bound Arf GTPases promote eukaryotic cell membrane trafficking and cytoskeletal remodelling. Arf activation is accelerated by guanine nucleotide-exchange factors (GEFs) using the critical catalytic glutamate in all known Sec7 domain sequences. Yeast Arf3p, a homologue of mammalian Arf6, is required for yeast invasive responses to glucose depletion. Here we identify Snf1p as a GEF that activates Arf3p when energy is limited. SNF1 is the yeast homologue of AMP-activated protein kinase (AMPK), which is a key regulator of cellular energy homeostasis. As activation of Arf3p does not depend on the Snf1p kinase domain, assay of regulatory domain fragments yield evidence that the C-terminal hydrophobic α-helix core of Snf1p is a non-canonical GEF for Arf3p activation. Thus, our study reveals a novel mechanism for regulating cellular responses to energy deprivation, in particular invasive cell growth, through direct Arf activation by Snf1/AMPK.

No MeSH data available.


Related in: MedlinePlus