A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters

Abstract

Cells rapidly remodel their proteomes to align their cellular metabolism to environmental conditions. Ubiquitin E3 ligases enable this response, by facilitating rapid and reversible changes to protein stability, localization, or interaction partners. In Saccharomyces cerevisiae, the GID E3 ligase regulates the switch from gluconeogenic to glycolytic conditions through induction and incorporation of the substrate receptor subunit Gid4, which promotes the degradation of gluconeogenic enzymes. Here, we show an alternative substrate receptor, Gid10, which is induced in response to changes in temperature, osmolarity, and nutrient availability, regulates the ART‐Rsp5 ubiquitin ligase pathway, a component of plasma membrane quality control. Proteomic studies reveal that the levels of the adaptor protein Art2 are elevated upon GID10 deletion. A crystal structure shows the basis for Gid10‐Art2 interactions, and we demonstrate that Gid10 directs a GID E3 ligase complex to ubiquitinate Art2. Our data suggest that the GID E3 ligase affects Art2‐dependent amino acid transport. This study reveals GID as a system of E3 ligases with metabolic regulatory functions outside of glycolysis and gluconeogenesis, controlled by distinct stress‐specific substrate receptors.

Structural and Functional Insights into GID/CTLH E3 Ligase Complexes

“…A paradigm has emerged of activation of the complex in response to a stimulus, causing the ubiquitination of targets that induces a biological change or cellular adaptation, followed by inactivation of the complex when it is no longer needed ( Figure 6 ). In S. cerevisiae , complex activation includes the induction of substrate receptors Gid4 and Gid10 during cellular stress, which is then followed by their own proteasomal degradation [ 10 , 39 , 73 ]. In D. melanogaster , translational upregulation of the UBE2H orthologue, the E2-conjugating enzyme for the complex, during MZT, activates the complex to ubiquitinate its substrates at a precise time [ 45 , 96 ].…”

“…Despite the sequence diversity in GID4 degron binding preferences observed using in vitro experiments, no GID4 substrate has currently been definitively determined outside of budding yeast. Slight structural differences in S. cerevisiae Gid10′s β-barrel enable the binding of a bulky hydrophobic residue in position 2 of the degron (as opposed to smaller Gly/Ala preferred for GID4), such as for its only known target thus far, Art2 (Nt-Pro-Phe-Ile-Thr) [ 36 , 37 , 39 ]. Gid11, the third S. cerevisiae interchangeable substrate receptor, recognizes proteins with an N-terminal Thr [ 13 ].…”

“…Unlike Gid4, Gid10 is not expressed under normal conditions or during glucose recovery, but is induced by heat shock, osmotic stress, or starvation of nitrogen or individual amino acids [ 12 , 14 , 39 ]. During heat shock, the N-terminal proline containing Art2 is a target of GID SR10 , its first one identified [ 39 ]. The regulation of Art2 by GID SR10 during heat shock in part affects the Rsp5-dependent import and degradation of amino acid transporters Lyp1 and Can1.…”

“…Gid10 induction by various stressors is only temporary: Gid10, similarly to Gid4 during glucose recovery, is a target of the GID complex itself and is quickly degraded [ 39 , 73 ]. The purpose of the negative autoregulation is seemingly to have the complex available to whichever substrate receptor is induced by distinct environmental perturbations, thus poised to quickly maintain homeostasis.…”

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import

“…It thereby recognizes specific N-terminal proline motifs in their sequence ( Chen et al, 2017 ). The substrate spectra and recognition motifs of the other specifying factors Gid10 ( Melnykov et al, 2019 ; Langlois et al, 2022 ) and Gid11 ( Kong et al, 2021 ) are less well understood.…”