Shevade Aishwarya, Strogolova Vera, Orlova Marianna, Yeo Chay Teng, Kuchin Sergei
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA.
mSphere. 2018 Jan 10;3(1). doi: 10.1128/mSphere.00482-17. eCollection 2018 Jan-Feb.
Snf1 protein kinase of the yeast is a member of the highly conserved eukaryotic AMP-activated protein kinase (AMPK) family, which is involved in regulating responses to energy limitation. Under conditions of carbon/energy stress, such as during glucose depletion, Snf1 is catalytically activated and enriched in the nucleus to regulate transcription. Snf1 catalytic activation requires phosphorylation of its conserved activation loop threonine (Thr210) by upstream kinases. Catalytic activation is also a prerequisite for Snf1's subsequent nuclear enrichment, a process that is mediated by Gal83, one of three alternate β-subunits of the Snf1 kinase complex. We previously reported that the mitochondrial voltage-dependent anion channel (VDAC) proteins Por1 and Por2 play redundant roles in promoting Snf1 catalytic activation by Thr210 phosphorylation. Here, we show that the Δ mutation alone, which by itself does not affect Snf1 Thr210 phosphorylation, causes defects in Snf1 and Gal83 nuclear enrichment and Snf1's ability to stimulate transcription. We present evidence that Por1 promotes Snf1 nuclear enrichment by promoting the nuclear enrichment of Gal83. Overexpression of Por2, which is not believed to have channel activity, can suppress the localization and transcription activation defects of the Δ mutant, suggesting that the regulatory role played by Por1 is separable from its channel function. Thus, our findings expand the positive roles of the yeast VDACs in carbon/energy stress signaling upstream of Snf1. Since AMPK/Snf1 and VDAC proteins are conserved in evolution, our findings in yeast may have implications for AMPK regulation in other eukaryotes, including humans. AMP-activated protein kinases (AMPKs) sense energy limitation and regulate transcription and metabolism in eukaryotes from yeast to humans. In mammals, AMPK responds to increased AMP-to-ATP or ADP-to-ATP ratios and is implicated in diabetes, heart disease, and cancer. Mitochondria produce ATP and are generally thought to downregulate AMPK. Indeed, some antidiabetic drugs activate AMPK by affecting mitochondrial respiration. ATP release from mitochondria is mediated by evolutionarily conserved proteins known as voltage-dependent anion channels (VDACs). One would therefore expect VDACs to serve as negative regulators of AMPK. However, our experiments in yeast reveal the existence of an opposite relationship. We previously showed that VDACs Por1 and Por2 positively regulate AMPK/Snf1 catalytic activation. Here, we show that Por1 also plays an important role in promoting AMPK/Snf1 nuclear localization. Our counterintuitive findings could inform research in areas ranging from diabetes to cancer to fungal pathogenesis.
酵母的Snf1蛋白激酶是高度保守的真核生物AMP激活蛋白激酶（AMPK）家族的成员，该家族参与调节对能量限制的反应。在碳/能量应激条件下，例如在葡萄糖耗尽期间，Snf1被催化激活并在细胞核中富集以调节转录。Snf1的催化激活需要上游激酶将其保守的激活环苏氨酸（Thr210）磷酸化。催化激活也是Snf1随后核富集的先决条件，这一过程由Snf1激酶复合体的三个交替β亚基之一Gal83介导。我们之前报道过，线粒体电压依赖性阴离子通道（VDAC）蛋白Por1和Por2在通过Thr210磷酸化促进Snf1催化激活方面发挥冗余作用。在这里，我们表明单独的Δ突变本身并不影响Snf1 Thr210磷酸化，但会导致Snf1和Gal83核富集以及Snf1刺激转录能力的缺陷。我们提供的证据表明Por1通过促进Gal83的核富集来促进Snf1的核富集。不被认为具有通道活性的Por2的过表达可以抑制Δ突变体的定位和转录激活缺陷，这表明Por1发挥的调节作用与其通道功能是可分离的。因此，我们的发现扩展了酵母VDACs在Snf1上游碳/能量应激信号传导中的积极作用。由于AMPK/Snf1和VDAC蛋白在进化中是保守的，我们在酵母中的发现可能对包括人类在内的其他真核生物中的AMPK调节有影响。AMP激活蛋白激酶（AMPKs）感知能量限制并调节从酵母到人类的真核生物中的转录和代谢。在哺乳动物中，AMPK对AMP与ATP或ADP与ATP比率的增加做出反应，并与糖尿病、心脏病和癌症有关。线粒体产生ATP，通常被认为会下调AMPK。事实上，一些抗糖尿病药物通过影响线粒体呼吸来激活AMPK。线粒体释放ATP是由被称为电压依赖性阴离子通道（VDACs）的进化保守蛋白介导的。因此，人们会期望VDACs作为AMPK的负调节因子。然而，我们在酵母中的实验揭示了一种相反的关系。我们之前表明VDACs Por1和Por2正向调节AMPK/Snf1催化激活。在这里，我们表明Por1在促进AMPK/Snf1核定位方面也起着重要作用。我们这一违反直觉的发现可能会为从糖尿病到癌症再到真菌发病机制等领域的研究提供信息。
