Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov;13(11):1522-31.
doi: 10.1111/j.1600-0854.2012.01406.x. Epub 2012 Sep 7.

Metabolic activation of the HOG MAP kinase pathway by Snf1/AMPK regulates lipid signaling at the Golgi

Hailan Piao  1 , John MacLean Freed, Peter Mayinger
Affiliations

Metabolic activation of the HOG MAP kinase pathway by Snf1/AMPK regulates lipid signaling at the Golgi

Hailan Piao et al. Traffic. 2012 Nov.

Abstract

Phosphatidylinositol-4-phosphate (PI(4)P) is an important regulator of Golgi function. Metabolic regulation of Golgi PI(4)P requires the lipid phosphatase Sac1 that translocates between endoplasmic reticulum (ER) and Golgi membranes. Localization of Sac1 responds to changes in glucose levels, yet the upstream signaling pathways that regulate Sac1 traffic are unknown. Here, we report that mitogen-activated protein kinase (MAPK) Hog1 transmits glucose signals to the Golgi and regulates localization of Sac1. We find that Hog1 is rapidly activated by both glucose starvation and glucose stimulation, which is independent of the well-characterized response to osmotic stress but requires the upstream element Ssk1 and is controlled by Snf1, the yeast homolog of AMP-activated kinase (AMPK). Elimination of either Hog1 or Snf1 slows glucose-induced translocation of Sac1 lipid phosphatase from the Golgi to the ER and thus delays PI(4)P accumulation at the Golgi. We conclude that a novel cross-talk between the HOG pathway and Snf1/AMPK is required for the metabolic control of lipid signaling at the Golgi.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Genetic interactions between sac1 and hog1 mutants. (A) Cells were plated in 5-fold serial dilutions (starting density 107 cells/ml) on rich growth medium (YPD) or on YPD supplemented with 1 M sorbitol or 1 M NaCl. (B-D) Cell growth rates of wild-type cells and HOG pathway mutants in wild-type or sac1Δ backgrounds. Growth rates at 30°C were simultaneously monitored by measuring OD600 every 10 min. (E) Osmosensitive activation of Hog1 via two parallel mechanisms downstream of Sho1 and Sln1. Factors displaying negative genetic interactions with sac1Δ mutants are highlighted in orange.
Figure 2
Figure 2
Glucose-dependent retrograde trafficking of Sac1 requires Hog1 MAPK. Localization of GFP-Sac1p in wild-type (A) and hog1Δ (B) strains. Strains were exponentially grown in SD-Ura (Exp), starved for glucose for 30 min (−Glu) and subsequently stimulated by glucose addition for 30 min (+Glu). Intracellular localization of GFP-Sac1 was determined by fluorescence microscopy. (C) Quantification of the distribution of GFP-Sac1 between ER and Golgi in wild-type and hog1Δ cells (200 cells/each growth condition; GFP-Sac1, n=3 +/− SD). D) Strains were exponentially grown in SD-Ura (Exp), starved for glucose for 30 min (−Glu) and subsequently subjected to 0.4 M NaCl (−Glu+NaCl). Intracellular localization of GFP-Sac1 was determined by fluorescence microscopy. Scale bars (A, B and D), 4 µm.
Figure 3
Figure 3
hog1Δ cells show delayed recovery of Golgi PI(4)P after glucose stimulation. Localization of the PI(4)P probe FAPP1-PH-GFP in (A) wild-type and (B) hog1Δ strains. The cells were cultivated in glucose-deprived conditions for 30 min and then stimulated with glucose and examined by fluorescence microscopy at the indicated time points. FAPP1-PH-GFP was expressed from a CEN-based plasmid. Scale bars (A, B), 4 µm
Figure 4
Figure 4
Glucose starvation-specific activation of the HOG pathway. (A) Time course of Hog1 phosphorylation in response to glucose starvation. Strains were exponentially grown in SC (Exp) and then starved for glucose (−Glu). Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting. B) Nuclear translocation of Hog1-GFP after glucose starvation. A strain expressing Hog1-GFP from a genomic copy was cultivated in YPD and then starved for glucose. At the indicated times Hog1-GFP localization and nuclear DAPI signals were monitored using fluorescence microscopy. Scale bar (B), 4 µm.
Figure 5
Figure 5
Specificity of metabolic Hog1 activation. (A) Hog1 phosphorylation is not induced by nitrogen starvation or rapamycin. Strains were exponentially grown in SC (Exp) and then starved for ammonium sulfate or treated with 0.5 or 1 µg/ml rapamycin. Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting. (B) Starvation- and osmotic stress-induced Hog1 phosphorylation in wild-type, ssk1Δ and sho1Δ strains. Yeast cells were exponentially grown in SC (Exp), subjected to 0.4 M NaCl (+NaCl) or starved for glucose for 30 min (−Glu). Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting. (C) Starvation-induced hyperphosphorylation of Hog1 in a sln1Δ strain. Strains were exponentially grown in SC (Exp) and then starved for glucose for 30 min (−Glu). Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting.
Figure 6
Figure 6
Glucose stimulation induces nuclear accumulation and transient Golgi localization of Hog1-GFP. Time course of Hog1 phosphorylation in response to glucose starvation and stimulation in wild-type (A) and ssk1Δ cells (B). Strains were exponentially grown in SC (Exp), starved for glucose for 30 min (−Glu) and then stimulated with glucose (+Glu). Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting. (C) Time course of Hog1-GFP localization in response to glucose stimulation. Strains expressing Hog1-GFP (green) and Sec7-RFP (red) from genomic copies were starved for glucose for 30 min and then stimulated with glucose and examined by fluorescence microscopy. Colocalization of Hog1-GFP and Sec7-RFP at punctate structures is indicated with arrows in the merged images. Scale bars (C), 2 µm.
Figure 7
Figure 7
Snf1 is essential for glucose regulation of Hog1 MAPK and Sac1 traffic (A) Starvation-induced phosphorylation of Hog1 in wild-type and snf1Δ cells. Strains were exponentially grown in SC (Exp) and then starved for glucose for 30 min (−Glu). Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting. (B) Time course of Hog1 phosphorylation in response to glucose starvation and stimulation in wild-type and snf1Δ cells. Strains were exponentially grown in YPD (Exp) and starved for glucose for 30 min (−Glu) and then stimulated with glucose (+Glu). Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting. (C, D) Localization of GFP-Sac1 in wild-type (C) and snf1Δ (D) cells. Strains were exponentially grown in SD-Ura (Exp), starved for glucose for 30 min (−Glu) and then stimulated by glucose addition for 30 min (+Glu). Intracellular localization of GFP-Sac1 was determined by fluorescence microscopy. (E) Quantification of the distribution of GFP-Sac1 between ER and Golgi in wild-type and hog1Δ cells (100 cells/each growth condition; GFP-Sac1, n=3 +/− SD). F) Stimulation of Hog1p phosphorylation via combretastatin-induced Snf1p activation. Strains were exponentially grown in SC (Exp) and then mock-treated or treated with 300 µM combretastatin A4 (CA4). Equal amounts of yeast extracts based on total protein content were analyzed by SDS-PAGE. Levels of Hog1 and phosphorylated Hog1 (Hog1-P) were examined by immunoblotting. Scale bars, (C, D), 4 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mager WH, Planta RJ Biochemisch Laboratorium VUATN. Coordinate expression of ribosomal protein genes in yeast as a function of cellular growth rate. Mol Cell Biochem. 1991;104(1–2):181–187. - PubMed
    1. Schmelzle T, Hall MN. Tor, a central controller of cell growth. Cell. 2000;103:253–262. - PubMed
    1. Jorgensen P, Rupes I, Sharom JR, Schneper L, Broach JR, Tyers M. A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev. 2004;18:2491–2505. - PMC - PubMed
    1. Novick P, Schekman R. Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979;76:1858–1862. - PMC - PubMed
    1. Anastasia SD, Nguyen DL, Thai V, Meloy M, Macdonough T, Kellogg DR. A link between mitotic entry and membrane growth suggests a novel model for cell size control. J Cell Biol. 2012;197:89–104. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources