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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 regulatory domain of Snf1p activates Arf3p.(a–d) Activation of Arf3p by Snf1p. [3H]GDP dissociation from and [35S]GTPγS binding to Arf3-dN17 in the presence of Snf1p immunoprecipitated from wild-type and yel1Δ cells (a,b), or recombinant Yel1-Sec7, Snf1-N and Snf1-C (c,d), were monitored by measuring radioactivity. The data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Active forms of Arf3p were precipitated by GST-Afi1N in snf1Δ cells expressing SNF1, SNF1-N or SNF1-C. Right panel, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to vector (Vec) control. **P<0.01; Student's t-test.
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f3: C-terminal regulatory domain of Snf1p activates Arf3p.(a–d) Activation of Arf3p by Snf1p. [3H]GDP dissociation from and [35S]GTPγS binding to Arf3-dN17 in the presence of Snf1p immunoprecipitated from wild-type and yel1Δ cells (a,b), or recombinant Yel1-Sec7, Snf1-N and Snf1-C (c,d), were monitored by measuring radioactivity. The data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Active forms of Arf3p were precipitated by GST-Afi1N in snf1Δ cells expressing SNF1, SNF1-N or SNF1-C. Right panel, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to vector (Vec) control. **P<0.01; Student's t-test.

Mentions: To test whether Snf1p possesses GEF activity, we first examined the guanine nucleotide-exchange activity of immuno-isolated Snf1p-HA from wild-type versus yel1Δ cells, and found that they both increased GDP dissociation from and GTPγS binding to recombinant Arf3p (Fig. 3a,b). We also purified recombinant Snf1 fragments to examine their GEF activity towards recombinant Arf3p in vitro, and found that Snf1-C, but not Snf1-N, catalysed the GDP release from and GTPγS loading of Arf3, which was comparable to the effect of the Sec7 domain of Yel1p (the currently known Arf3p GEF; Fig. 3c,d). Furthermore, Snf1-C did not interact with or accelerate the guanine nucleotide exchange of other Arf or Arf-like proteins, including Arf1, Arl1 and Arl3 (Supplementary Fig. 6a–c). Thus, the regulatory domain of Snf1 is a specific Arf3 GEF in vitro. Next, to examine whether Snf1p-C is sufficient to activate Arf3p in vivo, we overexpressed SNF1-C in snf1Δ cells and found that SNF1-C, but not SNF1-N, could activate Arf3p significantly (Fig. 3e). However, SNF1-C could not rescue the snf1Δ growth defect on nutrient-limited medium (Supplementary Fig. 2c). This result is expected considering that Snf1p/AMPK should have many downstream targets, in addition to Arf3p, in mediating its role as a central coordinator of cellular energy homeostasis.


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 activates Arf3p.(a–d) Activation of Arf3p by Snf1p. [3H]GDP dissociation from and [35S]GTPγS binding to Arf3-dN17 in the presence of Snf1p immunoprecipitated from wild-type and yel1Δ cells (a,b), or recombinant Yel1-Sec7, Snf1-N and Snf1-C (c,d), were monitored by measuring radioactivity. The data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Active forms of Arf3p were precipitated by GST-Afi1N in snf1Δ cells expressing SNF1, SNF1-N or SNF1-C. Right panel, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to vector (Vec) control. **P<0.01; Student's t-test.
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Related In: Results  -  Collection

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f3: C-terminal regulatory domain of Snf1p activates Arf3p.(a–d) Activation of Arf3p by Snf1p. [3H]GDP dissociation from and [35S]GTPγS binding to Arf3-dN17 in the presence of Snf1p immunoprecipitated from wild-type and yel1Δ cells (a,b), or recombinant Yel1-Sec7, Snf1-N and Snf1-C (c,d), were monitored by measuring radioactivity. The data are reported as the means±s.d. of the percentages of dissociated [3H]GDP and of bound [35S]GTPγS (n=3). (e) Active forms of Arf3p were precipitated by GST-Afi1N in snf1Δ cells expressing SNF1, SNF1-N or SNF1-C. Right panel, quantitative analysis of active Arf3p. Data are reported as the mean±s.d. of three experiments relative to vector (Vec) control. **P<0.01; Student's t-test.
Mentions: To test whether Snf1p possesses GEF activity, we first examined the guanine nucleotide-exchange activity of immuno-isolated Snf1p-HA from wild-type versus yel1Δ cells, and found that they both increased GDP dissociation from and GTPγS binding to recombinant Arf3p (Fig. 3a,b). We also purified recombinant Snf1 fragments to examine their GEF activity towards recombinant Arf3p in vitro, and found that Snf1-C, but not Snf1-N, catalysed the GDP release from and GTPγS loading of Arf3, which was comparable to the effect of the Sec7 domain of Yel1p (the currently known Arf3p GEF; Fig. 3c,d). Furthermore, Snf1-C did not interact with or accelerate the guanine nucleotide exchange of other Arf or Arf-like proteins, including Arf1, Arl1 and Arl3 (Supplementary Fig. 6a–c). Thus, the regulatory domain of Snf1 is a specific Arf3 GEF in vitro. Next, to examine whether Snf1p-C is sufficient to activate Arf3p in vivo, we overexpressed SNF1-C in snf1Δ cells and found that SNF1-C, but not SNF1-N, could activate Arf3p significantly (Fig. 3e). However, SNF1-C could not rescue the snf1Δ growth defect on nutrient-limited medium (Supplementary Fig. 2c). This result is expected considering that Snf1p/AMPK should have many downstream targets, in addition to Arf3p, in mediating its role as a central coordinator of cellular energy homeostasis.

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