Ubiquitin release from eL40 is required for cytoplasmic maturation and function of 60S ribosomal subunits in Saccharomyces cerevisiae

Abstract

Ubiquitin is generated by proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a linear polyubiquitin protein of head‐to‐tail monomers, or as a single N‐terminal moiety to one of two ribosomal proteins, eL40 (Ubi1/2 precursors) and eS31 (Ubi3 precursor). It has been proposed that the ubiquitin moiety fused to these ribosomal proteins could act as a chaperone by facilitating their efficient production, folding and ribosome assembly in Saccharomyces cerevisiae. We have previously shown that ubiquitin release from eS31 is required for yeast viability and that noncleaved Ubi3 can get incorporated into translation‐competent 40S subunits. In this study, we have analysed the effects of mutations that partially or totally impair cleavage of the ubiquitin‐eL40A fusion protein. While noncleaved Ubi1 is not able to support growth when it is the sole cellular source of eL40, it can assemble into nascent pre‐60S particles. However, Ubi1‐containing 60S ribosomal subunits are not competent for translation. This is likely due to a steric interference of the unprocessed ubiquitin with the binding and function of factors that interact with the ribosome's GTPase‐associated centre. In agreement with this suggestion, Ubi1‐containing ribosomes affect the efficient recycling of the anti‐association factor Tif6 and have a reduced presence of translation elongation factors. We conclude that the removal of the ubiquitin moiety from ribosomal protein eL40 is an essential prerequisite for both the cytoplasmic maturation and the functionality of 60S ribosomal subunits.

The Ubiquitin Moiety of Ubi1 Is Required for Productive Expression of Ribosomal Protein eL40 in Saccharomyces cerevisiae

“…Under wild-type conditions, the Ubi1/2 and Ubi3 precursor proteins could so far never be detected, suggesting that their proteolytic maturation occurs very rapidly, likely co-translationally, and consequently before the assembly of the respective r-proteins into pre-ribosomal particles [14,31,35]. We have previously studied the consequences of introducing mutations in the intersection between ubiquitin and eS31 within the Ubi3 precursor and between ubiquitin and eL40 within the Ubi1 precursor that partially or totally impair ubiquitin removal [35,36]. The obtained results indicate that the presence of ubiquitin hinders the assembly of the respective r-proteins into pre-ribosomal particles; therefore, assembly of either eS31 or eL40A is favored over that of non-cleaved Ubi3 or Ubi1, respectively [35,36].…”

“…We have previously studied the consequences of introducing mutations in the intersection between ubiquitin and eS31 within the Ubi3 precursor and between ubiquitin and eL40 within the Ubi1 precursor that partially or totally impair ubiquitin removal [35,36]. The obtained results indicate that the presence of ubiquitin hinders the assembly of the respective r-proteins into pre-ribosomal particles; therefore, assembly of either eS31 or eL40A is favored over that of non-cleaved Ubi3 or Ubi1, respectively [35,36]. Moreover, non-cleaved Ubi3 or Ubi1 variants confer lethal phenotypes when expressed as the sole source of eS31 or eL40, respectively.…”

“…Moreover, non-cleaved Ubi3 or Ubi1 variants confer lethal phenotypes when expressed as the sole source of eS31 or eL40, respectively. Only in these circumstances (i.e., upon depletion of the wild-type eS31 or eL40 counterparts), these mutant proteins get incorporated into nascent pre-ribosomal particles [35,36]. Strikingly, while Ubi3-containing r-particles can quite efficiently engage in translation [35], Ubi1-containing r-particles are delayed in their cytoplasmic maturation (i.e., Tif6 recycling), prevent r-subunit joining, and interfere with translation elongation [36].…”

Ribosomal Protein L40e Fused With a Ubiquitin Moiety Is Essential for the Vegetative Growth, Morphological Homeostasis, Cell Cycle Progression, and Pathogenicity of Cryptococcus neoformans

“…In eukaryotes, ubiquitin is usually expressed as a hybrid protein with a fused ribosomal protein, such as Rpl40e or Rps31e ( Clague and Urbé, 2010 ). The ubiquitin moiety could act as a molecular chaperone to the ribosomal proteins by facilitating their efficient production, folding, and ribosome assembly ( Lacombe et al, 2009 ; Martín-Villanueva et al, 2019 , 2020 ). Expression of ubi1 Δ ub -HA, as the only source of Rpl40e, caused a severe slow-growth phenotype and aggregation of ribosomal proteins in S. cerevisiae ( Martín-Villanueva et al, 2019 ).…”

“…In Saccharomyces cerevisiae , the ribosomal protein moiety is required for 60S or 40S subunit production, subunit assembly, and subsequent translation ( Finley et al, 1989 ; Fernández-Pevida et al, 2012 ). The ubiquitin moiety acts as a chaperone by facilitating efficient ribosome biogenesis ( Lacombe et al, 2009 ; Martín-Villanueva et al, 2019 , 2020 ). The ubi1 Δ or ubi2 Δ mutant shows a slow-growth phenotype while UBI3 deletion or double deletions of UBI1 and UBI2 cause a lethal phenotype in S. cerevisiae ( Finley et al, 1989 ).…”