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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.


Snf1p regulates Arf3p activation independently of kinase activity in response to glucose depletion.(a) Yeast cells cultured in medium with glucose depletion (YP) or with glucose recovery (YPD) for 2 h were detected with GST-Afi1N 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 relative to wild type (WT) in YPD. ***P<0.001; Student's t-test. (b) Equal concentrations of the indicated Σ1278b yeast cells were spotted onto glucose-depleted (YP) plates and incubated for 16 h at 30 °C. Yeast invasion on YP plates was determined after rinsing with water. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. (c,d) Active forms of Arf3p were precipitated by GST-Afi1N in yel1Δ cells expressing SNF1 with YPD or YP treatment for 2 h (c) and in snf1Δ cells expressing SNF1 and SNF1K84R with YP treatment for 2 h (d). Quantitative analysis of active Arf3p is presented in the lower panels. Data are reported as the mean±s.d. of three experiments relative to (c) yel1Δ in YPD and (d) vector (Vec). **P<0.01 and ***P<0.001; Student's t-test.
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f1: Snf1p regulates Arf3p activation independently of kinase activity in response to glucose depletion.(a) Yeast cells cultured in medium with glucose depletion (YP) or with glucose recovery (YPD) for 2 h were detected with GST-Afi1N 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 relative to wild type (WT) in YPD. ***P<0.001; Student's t-test. (b) Equal concentrations of the indicated Σ1278b yeast cells were spotted onto glucose-depleted (YP) plates and incubated for 16 h at 30 °C. Yeast invasion on YP plates was determined after rinsing with water. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. (c,d) Active forms of Arf3p were precipitated by GST-Afi1N in yel1Δ cells expressing SNF1 with YPD or YP treatment for 2 h (c) and in snf1Δ cells expressing SNF1 and SNF1K84R with YP treatment for 2 h (d). Quantitative analysis of active Arf3p is presented in the lower panels. Data are reported as the mean±s.d. of three experiments relative to (c) yel1Δ in YPD and (d) vector (Vec). **P<0.01 and ***P<0.001; Student's t-test.

Mentions: Then, because our initial screening also identified a serine/threonine kinase Snf1p (for sucrose non-fermenting), the homologue of mammalian AMPK, as a binding partner of Arf3pT31N in response to glucose depletion, we next investigated the role of this observed interaction. First, we found that Snf1p is critical for glucose depletion-induced Arf3p activation (Fig. 1a), and, in comparison, that the glucose sensing G-protein-coupled receptor Gpr1p is dispensable (Supplementary Fig. 1e). Arf3p activity is tightly regulated by glucose in the growth medium, and glucose-sensitive Arf3p activation depends on the presence of Snf1p (Fig. 1a). Consistent with these findings, both Arf3p and Snf1p were also required for glucose depletion-induced agar invasion (Fig. 1b); however, deletion of SNF1, but not ARF3, in yeast showed a pronounced growth defect on raffinose medium (Supplementary Fig. 2a). Strikingly, Snf1p overexpression enhanced Arf3p activation in both wild-type and yel1Δ cells (Supplementary Fig. 3a and Fig. 1c). This result indicates that Snf1p activates Arf3p independent of Yel1p, the only known Arf3p GEF to date.


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)

Snf1p regulates Arf3p activation independently of kinase activity in response to glucose depletion.(a) Yeast cells cultured in medium with glucose depletion (YP) or with glucose recovery (YPD) for 2 h were detected with GST-Afi1N 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 relative to wild type (WT) in YPD. ***P<0.001; Student's t-test. (b) Equal concentrations of the indicated Σ1278b yeast cells were spotted onto glucose-depleted (YP) plates and incubated for 16 h at 30 °C. Yeast invasion on YP plates was determined after rinsing with water. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. (c,d) Active forms of Arf3p were precipitated by GST-Afi1N in yel1Δ cells expressing SNF1 with YPD or YP treatment for 2 h (c) and in snf1Δ cells expressing SNF1 and SNF1K84R with YP treatment for 2 h (d). Quantitative analysis of active Arf3p is presented in the lower panels. Data are reported as the mean±s.d. of three experiments relative to (c) yel1Δ in YPD and (d) vector (Vec). **P<0.01 and ***P<0.001; Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525183&req=5

f1: Snf1p regulates Arf3p activation independently of kinase activity in response to glucose depletion.(a) Yeast cells cultured in medium with glucose depletion (YP) or with glucose recovery (YPD) for 2 h were detected with GST-Afi1N 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 relative to wild type (WT) in YPD. ***P<0.001; Student's t-test. (b) Equal concentrations of the indicated Σ1278b yeast cells were spotted onto glucose-depleted (YP) plates and incubated for 16 h at 30 °C. Yeast invasion on YP plates was determined after rinsing with water. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. ***P<0.001; Student's t-test. (c,d) Active forms of Arf3p were precipitated by GST-Afi1N in yel1Δ cells expressing SNF1 with YPD or YP treatment for 2 h (c) and in snf1Δ cells expressing SNF1 and SNF1K84R with YP treatment for 2 h (d). Quantitative analysis of active Arf3p is presented in the lower panels. Data are reported as the mean±s.d. of three experiments relative to (c) yel1Δ in YPD and (d) vector (Vec). **P<0.01 and ***P<0.001; Student's t-test.
Mentions: Then, because our initial screening also identified a serine/threonine kinase Snf1p (for sucrose non-fermenting), the homologue of mammalian AMPK, as a binding partner of Arf3pT31N in response to glucose depletion, we next investigated the role of this observed interaction. First, we found that Snf1p is critical for glucose depletion-induced Arf3p activation (Fig. 1a), and, in comparison, that the glucose sensing G-protein-coupled receptor Gpr1p is dispensable (Supplementary Fig. 1e). Arf3p activity is tightly regulated by glucose in the growth medium, and glucose-sensitive Arf3p activation depends on the presence of Snf1p (Fig. 1a). Consistent with these findings, both Arf3p and Snf1p were also required for glucose depletion-induced agar invasion (Fig. 1b); however, deletion of SNF1, but not ARF3, in yeast showed a pronounced growth defect on raffinose medium (Supplementary Fig. 2a). Strikingly, Snf1p overexpression enhanced Arf3p activation in both wild-type and yel1Δ cells (Supplementary Fig. 3a and Fig. 1c). This result indicates that Snf1p activates Arf3p independent of Yel1p, the only known Arf3p GEF to date.

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.