Hal4 and Hal5 Protein Kinases Are Required for General Control of Carbon and Nitrogen Uptake and Metabolism

Abstract

The yeast protein kinases Sat4/Hal4 and Hal5 are required for the plasma membrane stability of the K ؉ transporter Trk1 and some amino acid and glucose permeases. The transcriptomic analysis presented here indicates alterations in the general control of the metabolism of both nitrogen and carbon. Accordingly, we observed reduced uptake of methionine and leucine in the hal4 hal5 mutant. This decrease correlates with activation of the Gcn2-Gcn4 pathway, as measured by expression of the lacZ gene under the control of the GCN4 promoter. However, with the exception of methionine biosynthetic genes, few amino acid biosynthetic genes are induced in the hal4 hal5 mutant, whereas several genes involved in amino acid catabolism are repressed. Concerning glucose metabolism, we found that this mutant exhibits derepression of respiratory genes in the presence of glucose, leading to an increased activity of mitochondrial enzymes, as measured by succinate dehydrogenase (SDH) activity. In addition, the reduced glucose consumption in the hal4 hal5 mutant correlates with a more acidic intracellular pH and with low activity of the plasma membrane H ؉ -ATPase. As a compensatory mechanism for the low glycolytic rate, the hal4 hal5 mutant overexpresses the HXT4 highaffinity glucose transporter and the hexokinase genes. These results indicate that the hal4 hal5 mutant presents defects in the general control of nitrogen and carbon metabolism, which correlate with reduced transport of amino acids and glucose, respectively. A more acidic intracellular pH may contribute to some defects of this mutant.

Reciprocal Regulation of Target of Rapamycin Complex 1 and Potassium Accumulation

“…However, we did not obtain any evidence that Sch9 is required for potassium uptake or accumulation, as the growth pattern of strains lacking SCH9 is not altered by external potassium concentrations and the rapamycin-dependent decrease in potassium accumulation was similar in the wild type and sch9 mutant. 6 Previous experiments examining the subcellular localization of the TORC1-responsive Gln3 transcription factor, which is regulated by Npr1, also support this finding (42). These authors report that in mutants lacking HAL4 or HAL5, which have lower internal potassium, Gln3 nuclear localization is reduced, whereas in mutants lacking PPZ1, which have higher intracellular potassium, Gln3 accumulates in the nucleus (4,40).…”

“…More specifically, this kinase has been shown to phosphorylate members of the ART family, and in the case of Art1 this phosphorylation reduces its activity as an Rsp5 adaptor, leading to an increase in the plasma membrane accumulation of amino acid permeases, such as Can1 and Mup1 (27). We previously reported that in strains lacking the protein kinases Hal4 and Hal5, several plasma membrane transport proteins are aberrantly accumulated in the vacuole (5,6). We tested whether the overexpression of NPR1 and the corresponding phospho-inhibition of ART proteins would improve the growth phenotypes observed for the hal4 hal5 mutant.…”

“…We chose this permease based on our previous observations that Mup1 stability is acutely responsive to loss of HAL4 and HAL5 and that this permease is regulated by Art1 (6,27). As shown in Fig.…”

“…This mutant is known to have reduced internal potassium via regulation of Trk1 and Trk2, but how these kinases regulate potassium homeostasis is still being defined. Our previous studies have demonstrated that in strains lacking the Hal4 and Hal5 kinases, Trk1 and several other nutrient transporters aberrantly accumulate in the vacuole in the absence of potassium supplementation (5,6).…”

“…Two functionally redundant protein kinases, Hal4 (Sat4) and Hal5, are positive regulators of the Trk1 transporter (4). More specifically, they regulate plasma membrane stability of the Trk1 transporter and other nutrient permeases, such as Can1, Mup1, Fur4, or Hxt1 (5,6) by an unknown mechanism. We have observed that upon removal of potassium supplementation, these ion and nutrient transporters aberrantly accumulate in the vacuole.…”