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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.


Related in: MedlinePlus

C-terminal RS of Snf1p interacts with and activates Arf3p.(a) Snf1p, Snf1p-A2, and Snf1p-A5 were immunoprecipitated (IP) with anti-HA antibodies, and the bound proteins were immunoblotted for the presence of Arf3p. (b) Arf3p-GTP forms were precipitated by GST-Afi1N in snf1Δ cells expressing Snf1p, Snf1p-A2 or Snf1p-A5. Below, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to a vector (Vec) control. *P<0.05 and **P<0.01; Student's t-test. (c,d) [3H]GDP dissociation (c) from and [35S]GTPγS binding (d) to Arf3 in the presence of Snf1-C, Snf1-C-A2 or Snf1-C-A5 were monitored by measuring radioactivity. Data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Σ1278b yeast cells containing a SNF1 deletion transformed with different forms of SNF1 (full length, A2 or A5) were spotted onto YP plates for 16 h to examine agar penetration. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. **P<0.01; Student's t-test. (f) A working model for Arf3p activation by Snf1p to mediate invasive growth in response to glucose depletion. Snf1p/AMPK utilizes its N-terminal kinase domain to regulate FLO11 gene transcription and its atypical Arf GEF at the C-terminal regulatory domain to promote Arf3p activation in response to glucose deprivation.
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f5: C-terminal RS of Snf1p interacts with and activates Arf3p.(a) Snf1p, Snf1p-A2, and Snf1p-A5 were immunoprecipitated (IP) with anti-HA antibodies, and the bound proteins were immunoblotted for the presence of Arf3p. (b) Arf3p-GTP forms were precipitated by GST-Afi1N in snf1Δ cells expressing Snf1p, Snf1p-A2 or Snf1p-A5. Below, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to a vector (Vec) control. *P<0.05 and **P<0.01; Student's t-test. (c,d) [3H]GDP dissociation (c) from and [35S]GTPγS binding (d) to Arf3 in the presence of Snf1-C, Snf1-C-A2 or Snf1-C-A5 were monitored by measuring radioactivity. Data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Σ1278b yeast cells containing a SNF1 deletion transformed with different forms of SNF1 (full length, A2 or A5) were spotted onto YP plates for 16 h to examine agar penetration. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. **P<0.01; Student's t-test. (f) A working model for Arf3p activation by Snf1p to mediate invasive growth in response to glucose depletion. Snf1p/AMPK utilizes its N-terminal kinase domain to regulate FLO11 gene transcription and its atypical Arf GEF at the C-terminal regulatory domain to promote Arf3p activation in response to glucose deprivation.

Mentions: To gain further insight into how Snf1 acts as an Arf GEF, we next dissected the regulatory domain of Snf1p into Snf1-C1 (amino acid (a.a.) 392–518) and Snf1-C2 (a.a. 515–633), which are regions that interact with the γ- and β-subunits of the kinase, respectively2122. We found that Snf1-C1 is sufficient to interact with Arf3T31N (Supplementary Fig. 7a). Snf1-C1 contains auto-inhibitory sequence (AIS) and regulatory sequence (RS) that have been reported to repress Snf1p kinase activity22. Although Snf1-C1 did not share sequence similarity with canonical Sec7 domains, sequence alignment of the C1 region of Snf1p/AMPK homologues from different eukaryotes revealed that this region is evolutionarily conserved (Supplementary Fig. 7b). To identify the interaction interface, we utilized alanine scanning to examine interactions within regions of the AIS and RS (Supplementary Fig. 7c). None of the alanine mutants in the AIS disrupted the interaction between Snf1p and Arf3p. In contrast, the RS mutant Snf1p-A5 (a.a. 520–524) showed defective binding to Arf3pT31N (Supplementary Fig. 8a and Fig. 5a). Consistent with its failure to bind to Arf3p, expression of SNF1-A5 in snf1Δ cells could neither activate Arf3p (Supplementary Fig. 8b and Fig. 5b) nor restore yeast invasion (Fig. 5e). Another mutant, SNF1-A2 (a.a. 505–509), also showed defective Arf3 GEF activity as measured by the GDP dissociation (Fig. 5c) and GTPγS binding (Fig. 5d) of Arf3, even though it could still interact with Arf3p (Fig. 5a). Similarly, SNF1-A2 could not support Arf3p activation (Supplementary Fig. 8b and Fig. 5b) and yeast invasive growth (Fig. 5e) in snf1Δ cells. However, expression of Snf1p-A2, unlike Snf1p-A5, did not rescue the raffinose hypersensitivity of snf1Δ cells (Supplementary Fig. 8c), even though both mutants could be phosphorylated in response to glucose depletion (Supplementary Fig. 8d). These results indicate that Snf1p-A2 may contain both the catalytic residues for Arf3p activation and the critical amino acids for its transcriptional regulatory activity. Taken together, these findings suggest that the C-terminal α-helix hydrophobic core of Snf1p is responsible for the Arf3p GEF activity that becomes activated during glucose depletion-induced yeast invasion.


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 RS of Snf1p interacts with and activates Arf3p.(a) Snf1p, Snf1p-A2, and Snf1p-A5 were immunoprecipitated (IP) with anti-HA antibodies, and the bound proteins were immunoblotted for the presence of Arf3p. (b) Arf3p-GTP forms were precipitated by GST-Afi1N in snf1Δ cells expressing Snf1p, Snf1p-A2 or Snf1p-A5. Below, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to a vector (Vec) control. *P<0.05 and **P<0.01; Student's t-test. (c,d) [3H]GDP dissociation (c) from and [35S]GTPγS binding (d) to Arf3 in the presence of Snf1-C, Snf1-C-A2 or Snf1-C-A5 were monitored by measuring radioactivity. Data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Σ1278b yeast cells containing a SNF1 deletion transformed with different forms of SNF1 (full length, A2 or A5) were spotted onto YP plates for 16 h to examine agar penetration. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. **P<0.01; Student's t-test. (f) A working model for Arf3p activation by Snf1p to mediate invasive growth in response to glucose depletion. Snf1p/AMPK utilizes its N-terminal kinase domain to regulate FLO11 gene transcription and its atypical Arf GEF at the C-terminal regulatory domain to promote Arf3p activation in response to glucose deprivation.
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getmorefigures.php?uid=PMC4525183&req=5

f5: C-terminal RS of Snf1p interacts with and activates Arf3p.(a) Snf1p, Snf1p-A2, and Snf1p-A5 were immunoprecipitated (IP) with anti-HA antibodies, and the bound proteins were immunoblotted for the presence of Arf3p. (b) Arf3p-GTP forms were precipitated by GST-Afi1N in snf1Δ cells expressing Snf1p, Snf1p-A2 or Snf1p-A5. Below, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to a vector (Vec) control. *P<0.05 and **P<0.01; Student's t-test. (c,d) [3H]GDP dissociation (c) from and [35S]GTPγS binding (d) to Arf3 in the presence of Snf1-C, Snf1-C-A2 or Snf1-C-A5 were monitored by measuring radioactivity. Data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Σ1278b yeast cells containing a SNF1 deletion transformed with different forms of SNF1 (full length, A2 or A5) were spotted onto YP plates for 16 h to examine agar penetration. The percentage of invasive cells was quantified as described in Methods section. Data are reported as the mean±s.d. of three experiments. **P<0.01; Student's t-test. (f) A working model for Arf3p activation by Snf1p to mediate invasive growth in response to glucose depletion. Snf1p/AMPK utilizes its N-terminal kinase domain to regulate FLO11 gene transcription and its atypical Arf GEF at the C-terminal regulatory domain to promote Arf3p activation in response to glucose deprivation.
Mentions: To gain further insight into how Snf1 acts as an Arf GEF, we next dissected the regulatory domain of Snf1p into Snf1-C1 (amino acid (a.a.) 392–518) and Snf1-C2 (a.a. 515–633), which are regions that interact with the γ- and β-subunits of the kinase, respectively2122. We found that Snf1-C1 is sufficient to interact with Arf3T31N (Supplementary Fig. 7a). Snf1-C1 contains auto-inhibitory sequence (AIS) and regulatory sequence (RS) that have been reported to repress Snf1p kinase activity22. Although Snf1-C1 did not share sequence similarity with canonical Sec7 domains, sequence alignment of the C1 region of Snf1p/AMPK homologues from different eukaryotes revealed that this region is evolutionarily conserved (Supplementary Fig. 7b). To identify the interaction interface, we utilized alanine scanning to examine interactions within regions of the AIS and RS (Supplementary Fig. 7c). None of the alanine mutants in the AIS disrupted the interaction between Snf1p and Arf3p. In contrast, the RS mutant Snf1p-A5 (a.a. 520–524) showed defective binding to Arf3pT31N (Supplementary Fig. 8a and Fig. 5a). Consistent with its failure to bind to Arf3p, expression of SNF1-A5 in snf1Δ cells could neither activate Arf3p (Supplementary Fig. 8b and Fig. 5b) nor restore yeast invasion (Fig. 5e). Another mutant, SNF1-A2 (a.a. 505–509), also showed defective Arf3 GEF activity as measured by the GDP dissociation (Fig. 5c) and GTPγS binding (Fig. 5d) of Arf3, even though it could still interact with Arf3p (Fig. 5a). Similarly, SNF1-A2 could not support Arf3p activation (Supplementary Fig. 8b and Fig. 5b) and yeast invasive growth (Fig. 5e) in snf1Δ cells. However, expression of Snf1p-A2, unlike Snf1p-A5, did not rescue the raffinose hypersensitivity of snf1Δ cells (Supplementary Fig. 8c), even though both mutants could be phosphorylated in response to glucose depletion (Supplementary Fig. 8d). These results indicate that Snf1p-A2 may contain both the catalytic residues for Arf3p activation and the critical amino acids for its transcriptional regulatory activity. Taken together, these findings suggest that the C-terminal α-helix hydrophobic core of Snf1p is responsible for the Arf3p GEF activity that becomes activated during glucose depletion-induced yeast invasion.

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