Purification and characterization of the three Snf1-activating kinases of <i>Saccharomyces cerevisiae</i>

Elbing, Karin; McCartney, Rhonda R.; Schmidt, Martin C.

doi:10.1042/bj20051213

Cited by 68 publications

(84 citation statements)

References 43 publications

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“…The GST-tagged Snf1 kinase domain was purified from bacterial cells by affinity chromatography (21). The cDNA for human protein phosphatase (PP) 2C␣ was purchased from Open Biosystems (IHS1382-8646531) and inserted into the bacterial expression plasmid pET14b (Novagen).…”

Section: Methodsmentioning

confidence: 99%

“…Rabbit PP1 was purchased from Sigma (P7937). Yeast Glc7 was TAP-purified as described (21). Human AMPK composed of the ␣ 1 , ␤ 1 , and ␥ 1 subunits was purified as described (22).…”

Section: Methodsmentioning

confidence: 99%

“…Again, we detected statistically significant protection mediated by both AMP and ADP, with ADP showing much greater efficacy. We were unable to test ATP directly in this system because TAP-purified Snf1 contains low levels of the Snf1-activating kinase (21). However, we tested the ability of a non-hydrolyzable ATP analog, AMP-PNP, to mediate protection and found that the addition of AMP-PNP caused a slight but statistically significant level of protection.…”

Section: Adenylate-mediated Protection From Dephosphorylation-mentioning

confidence: 99%

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Subunit and Domain Requirements for Adenylate-mediated Protection of Snf1 Kinase Activation Loop from Dephosphorylation*

Chandrashekarappa¹,

McCartney²,

Schmidt³

2011

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

“…Rabbit PP1 was purchased from Sigma (P7937). Yeast Glc7 was TAP-purified as described (21). Human AMPK composed of the ␣ 1 , ␤ 1 , and ␥ 1 subunits was purified as described (22).…”

Section: Methodsmentioning

confidence: 99%

Section: Adenylate-mediated Protection From Dephosphorylation-mentioning

confidence: 99%

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Subunit and Domain Requirements for Adenylate-mediated Protection of Snf1 Kinase Activation Loop from Dephosphorylation*

Chandrashekarappa¹,

McCartney²,

Schmidt³

2011

Journal of Biological Chemistry

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“…Each of the protein kinases Sak1, Tos3, and Elm1 phosphorylates SNF1 in vivo (5,7). Sak1 is the primary upstream kinase (8,(51)(52)(53), and its C-terminal region interacts with the Snf1 kinase domain (43). To assess the role of the heterotrimer in determining interactions with activating kinases in vivo, we examined phosphorylation of Snf1(1-309).…”

Section: Mutations That Cause Phosphorylation Of the Heterotrimer Duringmentioning

confidence: 99%

Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases

Ruiz

Liu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The SNF1/AMP-activated protein kinases are αβγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the β and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking β-and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.

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“…The Snf1p kinase is required for pseudohyphal growth and for growth on secondary carbon sources (23)(24)(25). Snf1p is regulated by the kinase Sak1p during nitrogen signaling, and Snf1p pairs with its Gal83p ␤-subunit isoform to activate expression of downstream targets that enable pseudohyphal growth (26,27). Recent genomic studies have identified additional signaling pathways and gene sets that contribute to yeast pseudohyphal growth (28 -31), collectively indicating an extensive regulatory network controlling gene and protein activity during the filamentous growth transition.…”

mentioning

confidence: 99%

A Profile of Differentially Abundant Proteins at the Yeast Cell Periphery during Pseudohyphal Growth

Shively

Jin

et al. 2010

Journal of Biological Chemistry

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Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, we expect that the protein complement at the yeast cell periphery plays a critical and tightly regulated role in pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By isobaric tags for relative and absolute quantification chemistry and two-dimensional liquid chromatography-tandem mass spectrometry, we profiled 2463 peptides and 356 proteins, identifying 11 differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1. Pun1p abundance is doubled under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1⌬ mutants are noninvasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1⌬ mutants misregulate amino acid biosynthetic genes during nitrogen stress. By chromatin immunoprecipitation and reverse transcription-PCR, we find that the filamentous growth factor Mss11p directly binds the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of differential protein abundance during pseudohyphal growth, identifying a previously uncharacterized membrane compartment of Can1 protein required for wild-type nitrogen signaling and filamentous growth.Under conditions of nitrogen stress, certain strains of Saccharomyces cerevisiae implement a dramatic change in growth form characterized by the development of multicellular pseudohyphal filaments (1-4). During pseudohyphal growth, yeast cells delay in G 2 /M, resulting in an extended period of apically polarized growth and an elongated cell morphology (5-7). The yeast cells also exhibit an altered pattern of budding in which daughter cells emerge from mother cells predominantly opposite the birth end, as opposed to the bipolar pattern of bud emergence observed in diploid cells under conditions of vegetative growth (8). Perhaps most strikingly, filamentous yeast cells remain physically connected after cell division (1). The resulting pseudohyphal filaments adhere to and invade growth substrates, such as agar (9 -11). Thought to be a foraging mechanism, yeast filamentous growth has been studied extensively as a model for related hyphal growth transitions in the opportunistic human pathogen Candida albicans, wherein these growth transitions are required for virulen...

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Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae

Cited by 68 publications

References 43 publications

Subunit and Domain Requirements for Adenylate-mediated Protection of Snf1 Kinase Activation Loop from Dephosphorylation*

Subunit and Domain Requirements for Adenylate-mediated Protection of Snf1 Kinase Activation Loop from Dephosphorylation*

Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases

A Profile of Differentially Abundant Proteins at the Yeast Cell Periphery during Pseudohyphal Growth

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