F-Box Protein Grr1 Interacts with Phosphorylated Targets via the Cationic Surface of Its Leucine-Rich Repeat

Abstract

The flexibility and specificity of ubiquitin-dependent proteolysis are mediated, in part, by the E3 ubiquitin ligases. One class of E3 enzymes, SKp1/cullin/F-box protein (SCF), derives its specificity from F-box proteins, a heterogeneous family of adapters for target protein recognition. Grr1, the F-box component of SCF Grr1 , mediates the interaction with phosphorylated forms of the G 1 cyclins Cln1 and Cln2. We show that binding of Cln2 by SCF Grr1 was dependent upon its leucine-rich repeat (LRR) domain and its carboxy terminus. Our structural model for the Grr1 LRR predicted a high density of positive charge on the concave surface of the characteristic horseshoe structure. We hypothesized that specific basic residues on the predicted concave surface are important for recognition of phosphorylated Cln2. We show that point mutations that converted the basic residues on the concave surface but not those on the convex surface to neutral or acidic residues interfered with the capacity of Grr1 to bind to Cln2. The same mutations resulted in the stabilization of Cln2 and Gic2 and also in a spectrum of phenotypes characteristic of inactivation of GRR1, including hyperpolarization and enhancement of pseudohyphal growth. It was surprising that the same residues were not important for the role of Grr1 in nutrient-regulated transcription of HXT1 or AGP1. We concluded that the cationic nature of the concave surface of the Grr1 LRR is critical for the recognition of phosphorylated targets of SCF Grr1 but that other properties of Grr1 are required for its other functions.

PP2ACdc55regulates G1cyclin stability

“…grr1B4Q and grr1ΔC alleles have defects in binding to and degrading Cln2 17 and are synthetic lethal with loss of CDC55 (Fig. 3).…”

“…The grrR485Q allele has a substituted glutamine (Q) at residue R485, which resides on the convex surface of the LRR; this residue is not required for phosphorylated substrate binding. 17 The grr1ΔC allele has 234 C-terminal residues deleted and is defective in substrate binding. FOA s was used to test if cells harboring these alleles required PP2A Cdc55 for viability.…”

“…Thus, it was determined if Grr1 substrate binding was required for cdc55 cell viability. cdc55 cells were used harboring the grr1 alleles isolated by Hsuing et al, 17 which are able or unable to bind substrate.…”

“…15,16 The F-box protein Grr1 recognizes the Cdc28-dependent autophosphorylation of multiple Cln1/2 PEST domain sites, resulting in SCF Grr1 -phospho-Cln1/2 association. 12,[17][18][19] This autocatalytic targeting keeps Cln1/2 unstable throughout the cell cycle, including in pre-Start G 1 cells. 13 Interestingly, deletion of GRR1 causes the accumulation of Cln1 and Cln2 and an elongated phenotype.…”

“…13 Interestingly, deletion of GRR1 causes the accumulation of Cln1 and Cln2 and an elongated phenotype. 17 Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase comprised of two regulatory subunits (A and B) and a single catalytic subunit (C). [20][21][22][23] B subunits direct substrate specificity and subcellular location and are necessary for PP2A to interact with and dephosphorylate a variety of substrates.…”