The Snf1 protein kinase has many different roles in cellular responses to stress, and the presence of three different isoforms of the β subunit confers versatility on the kinase. We have examined the unique subcellular distribution exhibited by the Sip1 β subunit of the kinase and its regulation. We show that Sip1 relocalizes from the cytoplasm to the vacuolar periphery when glucose-grown cells are subjected to acute carbon stress and that the Snf1 catalytic subunit accompanies Sip1. In addition, Sip1 is found around the vacuole in a substantial fraction of cells during growth on carbon sources such as galactose and raffinose, which are less preferred than glucose. The N terminus of Sip1, which is divergent from the N termini of the other two β subunits, is responsible for its localization to the vacuolar periphery; moreover, the glycine of the N-myristoylation consensus sequence is required, suggesting that Sip1 associates directly with the vacuolar membrane. This relocalization may target Snf1 catalytic activity to substrates present at the vacuolar membrane; however, we cannot exclude the possibility that relocalization instead serves to sequester the Sip1 form of the kinase from targets elsewhere. The observed relocalization strongly suggests a role for Sip1 during carbon source transitions. Although glucose-grown sip1Δ mutant cells appeared to adapt normally to other carbon sources on solid and liquid medium (K. Hedbacker, unpublished results), functional redundancy among the β subunits may have obscured any defect.
We further present evidence that the PKA pathway regulates the localization of Sip1. In mutants lacking PKA, Sip1 was found constitutively around the vacuole, indicating that PKA is required to maintain the cytoplasmic localization of Sip1 during growth in glucose. Similar results were obtained with a mutant lacking two of the three PKA catalytic subunits and overexpressing cAMP phosphodiesterase, confirming that PKA activity is the relevant factor. Conversely, localization of Sip1 to the vacuole in response to carbon stress was impaired in a mutant lacking the Bcy1 regulatory subunit of PKA. The Snf1 catalytic subunit was also found at the vacuolar membrane in PKA-deficient cells during growth in glucose, indicating that PKA regulates the localization of Snf1-Sip1 protein kinase complexes.
PKA is known to have broad roles in cellular regulation, controlling such processes as cell growth, metabolism, stress resistance, and filamentous invasive growth (for reviews see references 30
, and 49
). The present findings establish a novel form of regulation by the PKA pathway, in which PKA regulates the localization of Snf1-Sip1. The possibility that PKA also regulates the catalytic activity of Snf1-Sip1 has not been excluded.
The synthesis of cAMP by adenylate cyclase is stimulated by Ras proteins and by glucose via the G protein-coupled receptor Gpr1 and the Gα protein Gpa2. It seems likely that the Gpr1-Gpa2 glucose-sensing pathway is involved in regulating the localization of Snf1-Sip1. The Gpr1 receptor is activated by glucose and interacts with Gpa2 to stimulate cAMP synthesis and to promote filamentous and invasive growth (4
). We note that Sip1 has a modest inhibitory role in haploid invasive growth (46
). Glucose-induced stimulation of cAMP synthesis also requires intracellular phosphorylation of the sugar (34
), and kelch repeat proteins that mimic Gβ subunits have been implicated in the signaling mechanism (10
Previous studies showed that the catalytic activity of Snf1 protein kinase is controlled by multiple regulatory inputs, including three upstream kinases (12
), protein phosphatase 1 (24
), and the Std1 protein (19
). It is now clear that localization of the kinase is also controlled by more than one mechanism. We present evidence that Sip1 and Gal83 respond to distinct signals: 2-deoxyglucose prevents nuclear localization of Gal83 but does not affect the localization of Sip1, and PKA regulates the localization of Sip1 but not that of Gal83. These findings are consistent with evidence that the activity of PKA toward Msn2 is insensitive to 2-deoxyglucose (8
) and with genetic evidence suggesting that glucose-6-phosphate is a candidate for the signal controlling the nucleocytoplasmic distribution of Gal83 (45
). Thus, these two β subunits exhibit distinct patterns of subcellular localization and their localization is regulated by different pathways. Localization affects the access of Snf1 to substrates, and regulation of localization by different pathways ensures access to different substrates in response to different signals. This complexity adds further versatility to Snf1 protein kinase function.