Mutational Analyses of the Cysteine-Rich Domain of Yvh1, a Protein Required for Translational Competency in Yeast.

Abstract

Simple SummaryRibosome maturation in eukaryotes is a highly energy-consuming, evolutionarily conserved process, directed by a large number of auxiliary proteins (>200) called trans-acting factors (TAFs). TAFs direct the ordered and directional assembly of immature pre-40S and pre-60S complexes in multiple cellular compartments, but they are not themselves components of translationally competent ribosomes. Although the enzymatic properties of many TAFs are understood, others assist ribosome maturation without predicted enzymatic activity. An important late-acting 60S TAF is Mrt4, which initially binds to pre-60S complexes within the nucleus that must be released from pre-60S complexes following translocation into in the cytoplasm prior to the addition of a complex of acidic phosphoproteins denoting the ribosomal stalk, an essential component of the peptidyl transferase center (PTC) of ribosomes. Prior genetic studies implicated the dual-specificity phosphatase Yvh1 as the protein responsible for cytoplasmic Mrt4 removal, but how Yvh1 facilitates this is unknown. yvh1Δ strains are viable but grow slowly due to translational defects associated with improper ribosome assembly. Yvh1-dependent Mrt4 removal is independent of YVH1’s protein phosphatase domain but instead maps to an evolutionarily conserved cysteine-rich domain (CRD) of unknown function. In order to better understand how YVH1’s CRD facilitates Mrt4 removal and thus 60S maturation, we identified loss-of-function (LOF) variants of Yvh1 incapable of displacing Mrt4 precluding the addition of the ribosomal stalk. This approach additionally identified a variant of Yvh1 (Yvh1F283L) that functions as an expression-dependent, dominant-negative variant capable of perturbing ribosome assembly in cells containing wild-type YVH1. These findings are consistent with, and expand on, recent structural models for Yvh1-dependent Mrt4 removal from pre-60S complexes and generate novel first-generation probes that can be used to better understand eukaryotic ribosomal maturation.Ribosome assembly is a complex biological process facilitated by >200 trans-acting factors (TAFs) that function as scaffolds, place-holders or complex remodelers to promote efficient and directional ribosomal subunit assembly but are not themselves part of functional ribosomes. One such yeast TAF is encoded by Mrt4 which assembles onto pre-60S complexes in the nuclear compartment and remains bound to pre-60S complexes as they are exported into the cytoplasm. There, Mrt4 is displaced from pre-60S complexes facilitating the subsequent addition of the ribosomal stalk complex (P0/P1/P2). Ribosomal stalk proteins interact with translational GTPases (trGTPase) which facilitate and control protein synthesis on the ribosome. The rRNA-binding domain of Mrt4 is structurally similar to P0, with both proteins binding to the same interface of pre-60S subunits in a mutually exclusive manner; the addition of the ribosomal stalk therefore requires the displacement of Mrt4 from pre-60S subunits. Mrt4 removal requires the C-terminal cysteine-rich domain (CRD) of the dual-specificity phosphatase Yvh1. Unlike many other TAFs, yeast lacking Yvh1 are viable but retain Mrt4 on cytoplasmic pre-60S complexes precluding ribosomal stalk addition. Although Yvh1’s role in Mrt4 removal is well established, how Yvh1 accomplishes this is largely unknown. Here, we report an unbiased genetic screen to isolate Yvh1 variants that fail to displace Mrt4 from pre-60S ribosomes. Bioorthogonal non-canonical amino acid tagging (BONCAT) approaches demonstrate that these YVH1 loss-of-function variants also display defects in nascent protein production. The further characterization of one LOF variant, Yvh1F283L, establishes it as an expression-dependent, dominant-negative variant capable of interfering with endogenous Yvh1 function, and we describe how this Yvh1 variant can be used as a novel probe to better understand ribosome maturation and potentially ribosome heterogeneity in eukaryotes.