The Rsp5 E3 Ligase Mediates Turnover of Low Affinity Phosphate Transporters in Saccharomyces cerevisiae

Abstract

In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho ؊ phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4⌬ mutant in the presence of limiting inorganic phosphate (P i ). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho ؉ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.The yeast Saccharomyces cerevisiae has evolved an elaborate system to sense, acquire, and store inorganic phosphate (P i ) in response to its availability in the extracellular environment (for review, see Refs. 1 and 2). The PHO system consists of (i) the Pho3, Pho5, Pho11, and Pho12 acid phosphatases that are localized to the periplasmic space, (ii) the Pho8 and Pho13 alkaline phosphatases that are localized to the vacuole and periplasm, respectively, (iii) the high affinity plasma membrane P i transporters Pho84 and Pho89, which are regulated in response to P i availability, and the low affinity, constitutively expressed P i transporters Pho87, Pho90, and Pho91, (iv) Git1, a transporter that scavenges glycerophosphoinositol derived from secreted phosphatidylinositol, thereby replenishing inositol, P i , and glycerol (3), and (v) the PHM proteins that are involved in the synthesis and breakdown of polyphosphate, a storage form of P i in the vacuole (for review, see Ref. 4).The PHO regulon is responsible for scavenging P i and has been most extensively studied with regard to regulation of PHO5 expression. The Pho84 transporter and the Pho80 -Pho85 cyclin/cyclin-dependent kinase are negative regulators of PHO5 transcription, whereas the Pho81 inhibitor of Pho80 -Pho85 and the Pho4 transcriptional activator are positive regulators. In the presence of high external concentrations of P i , Pho85 phosphorylates Pho4, resulting in Pho4 nuclear export via the Msn5 exportin and cytoplasmic retention such that PHO5 is repressed (5). Conversely, when P i concentrations are low, Pho81 inhibits Pho85 activity. Pho4 remains unphosphorylated and has high affinity for the Pse1 importin, resulting i...

Genome-wide Fitness Profiles Reveal a Requirement for Autophagy During Yeast Fermentation

“…When phosphate is plentiful, Pho84 is removed from the plasma membrane and targeted to the vacuole for degradation (Lagerstedt et al 2002). Similarly, Pho91 and another low-affinity phosphate transporter, Pho87, are targeted for endocytosis and vacuolar degradation by ubiquitination (Estrella et al 2008). As our synthetic grape juice medium was not limiting for phosphate, we speculate that unnecessary phosphate transport expression and recycling may actually confer a fitness disadvantage under these conditions.…”

Functional Interaction of the Ess1 Prolyl Isomerase with Components of the RNA Polymerase II Initiation and Termination Machineries

“…5, Glc is ϪUra medium containing 4% glucose, whereas Gal medium is ϪUra medium containing 2% galactose and 0.1% raffinose. High-and low-P i media contain potassium phosphate at a final concentration of either 1 mM or 5 M, respectively, prepared as described previously (11). Growth media for liquid ␤-galactosidase activities are described below; solid X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) indicator medium was prepared as described online (biochemistry.ucsf.edu/labs/herskowitz/ xgalagar.html).…”

Phosphate homeostasis in the yeast Saccharomyces cerevisiae, the key role of the SPX domain‐containing proteins

“…However, independently of the conditions tested, the vacuolar targeting of both Pho87 and Pho90 strictly required the presence of their N-terminal SPX domain. Moreover, another study performed by Estrella et al (2008) [47] also showed that Pho87 and Pho91, in response to Pi deficiency, were ubiquitinated in an E3 ubiquitin ligase Rsp5-dependent manner before being targeted for endocytosis and degraded in the vacuole [47]. Consequently, in order to shed further light on the mechanisms regulating the activity of Pho87, Pho90 and Pho91, a key step would be the complete identification of the mechanisms involved in the vacuolar targeting of Pho87 and Pho90, identifying their interacting partner proteins as well as defining the key role of the SPX domain in these processes.…”