Papers by Erik Joel Jonas Gutierrez
Nucleic acids research, Jan 16, 2017
Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid... more Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the p...
Critical reviews in biochemistry and molecular biology
In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular pro... more In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a β-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of...
Molecular Cell, 2014
Approximately 30% of eukaryotic proteins contain hydrophobic signals for localization to the secr... more Approximately 30% of eukaryotic proteins contain hydrophobic signals for localization to the secretory pathway. These proteins can be mislocalized in the cytosol due to mutations in their targeting signals, certain stresses, or intrinsic inefficiencies in their translocation. Mislocalized proteins (MLPs) are protected from aggregation by the Bag6 complex and degraded by a poorly characterized proteasomedependent pathway. Here, we identify the ubiquitin ligase RNF126 as a key component of the MLP degradation pathway. In vitro reconstitution and fractionation studies reveal that RNF126 is the primary Bag6-dependent ligase. RNF126 is recruited to the N-terminal Ubl domain of Bag6 and preferentially ubiquitinates juxtahydrophobic lysine residues on Bag6-associated clients. Interfering with RNF126 recruitment in vitro prevents ubiquitination, and RNF126 depletion in cells partially stabilizes a Bag6 client. Bag6-dependent ubiquitination can be recapitulated with purified components, paving the way for mechanistic analyses of downstream steps in this cytosolic quality control pathway.
Nature, 2011
A substantial proportion of the genome encodes membrane proteins that are delivered to the endopl... more A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways 1. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis 2,3. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here, we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone 4. Bag6 complex capture depends on unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 clients is transferred to TRC40 for membrane insertion, while the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex impairs efficient ubiquitination selectively of MLPs. Thus, by its presence on ribosomes synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling permits fast-tracking of MLPs for degradation without futile engagement of cytosolic folding machinery. Protein targeting and translocation into the ER are not perfectly efficient 5,6 , thereby necessitating pathways to degrade MLPs released inappropriately into the cytosol. For example, mammalian prion protein (PrP), a widely expressed GPI-anchored cell surface glycoprotein, displays ~5-15% translocation failure in vitro, in cells, and in animals 2,3,5-10. This non-translocated population of PrP is degraded efficiently by a proteasome-dependent pathway, limiting cytosolic PrP levels at steady state 2,3,9,10. Prompt degradation is essential Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Molecular Cell, 2013
Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stim... more Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.
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Papers by Erik Joel Jonas Gutierrez