Guanine nucleotide exchange in the ribosomal GTPase EFL1 is modulated by the protein mutated in the Shwachman–Diamond Syndrome

Gijsbers, Abril; García-Márquez, Adrián; Luviano, Axel; Sánchez-Puig, Nuria

doi:10.1016/j.bbrc.2013.06.077

Cited by 23 publications

(21 citation statements)

References 37 publications

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“…Previous results in our group suggested that the SBDS protein increased the affinity of EFL1 for GTP as evidenced by a decrease on its K m value (16). If SBDS acted as GTP-stabilizing factor or GSF for EFL1 it would only modify the binding to GTP but not to GDP.…”

Section: Resultsmentioning

confidence: 67%

“…In fact, the K d for the GTP analogue resembles that obtained for GDP in the presence of SBDS, 677 and 618 M, respectively, with the corresponding dissociation rate constants varying only 3-fold. The dissociation rate constants for the different GTP binding experiments were calculated from the y intercept of the apparent rate constant dependence on the nucleotide concentration and not directly from a dissociation experiment to avoid ambiguous results arising from the catalysis (k cat 0.4 min Ϫ1 ) (16). This had an impact on the fitting uncertainties because the k app for the rearrangement were almost independent of the nucleotide concentration at the attainable experimental conditions (20).…”

Section: Complexmentioning

confidence: 99%

“…The latter can be further subdivided in 1) factors that favor the release of bound GDP and 2) factors that stabilize the interaction with GTP (15). Recently, we have shown that the interaction of EFL1 with SBDS resulted in a decrease of the Michaelis-Menten (K m ) constant for GTP and thus SBDS acts as a GEF for EFL1 (16). However, that work did not address which binding constant the SBDS protein modifies.…”

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confidence: 99%

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Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein

García-Márquez

Gijsbers

Mora³

et al. 2015

Journal of Biological Chemistry

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Background: EFL1 and the protein mutated in the Shwachman-Diamond syndrome (SBDS) participate in the maturation of the 60S ribosomal subunit. Results: SBDS increases the dissociation constant of EFL1 for GDP 60-fold. SBDS S143L disease mutation disrupts the interaction with EFL1. Conclusion: SBDS favors GDP dissociation from EFL1 and disease mutations abolishe this function. Significance: SBDS disease mutations prevent the nucleotide exchange regulation on EFL1.

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Section: Resultsmentioning

confidence: 67%

Section: Complexmentioning

confidence: 99%

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confidence: 99%

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Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein

García-Márquez

Gijsbers

Mora³

et al. 2015

Journal of Biological Chemistry

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“…However, deletion of REI1 makes cells grow very slowly, and deletion of ARX1 relieves the growth defect of rei1⌬ cells (42). Mutations in the Tif6 release factors, Efl1 and Sdo1, also cause severe growth retardation, and mutant Tif6, having lower affinity for the ribosome, bypasses the requirement of Efl1 and Sdo1 and restores the growth rate (14,56). The genetic and cell biological evidence from this study suggests that abnormal retention of Puf6 is more deleterious than loss of Puf6 from the ribosome synthesis pathway.…”

Section: Discussionmentioning

confidence: 99%

The Roles of Puf6 and Loc1 in 60S Biogenesis Are Interdependent, and Both Are Required for Efficient Accommodation of Rpl43

Yang

Ting

Liang

et al. 2016

Journal of Biological Chemistry

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Puf6 and Loc1 have two important functional roles in the cells, asymmetric mRNA distribution and ribosome biogenesis. Puf6 and Loc1 are localized predominantly in the nucleolus. They bind ASH1 mRNA, repress its translation, and facilitate the transport to the daughter cells. Asymmetric mRNA distribution is important for cell differentiation. Besides their roles in mRNA localization, Puf6 and Loc1 have been shown to be involved in 60S biogenesis. In puf6⌬ or loc1⌬ cells, pre-rRNA processing and 60S export are impaired and 60S subunits are underaccumulated. The functional studies of Puf6 and Loc1 have been focused on ASH1 mRNA pathway, but their roles in 60S biogenesis are still not clear. In this study, we found that Puf6 and Loc1 interact directly with each other and both proteins interact with the ribosomal protein Rpl43 (L43e). Notably, the roles of Puf6 and Loc1 in 60S biogenesis are interdependent, and both are required for efficient accommodation of Rpl43. Loc1 is further required to maintain the protein level of Rpl43. Additionally, the recruitment of Rpl43 is required for the release of Puf6 and Loc1. We propose that Puf6 and Loc1 facilitate Rpl43 loading and are sequentially released from 60S after incorporation of Rpl43 into ribosomes in yeast.Almost every process in a cell is mediated by proteins, the products of translating mRNA by ribosomes. Ribosomes are composed of two subunits, a small subunit and a large subunit, 40S and 60S, respectively, in eukaryotic cells. In Saccharomyces cerevisiae, the 40S and 60S subunits are assembled from a large primary 35 S rRNA transcribed by RNA polymerase I. Processing of this rRNA yields 5.8S, 18S, and 25S rRNAs. The 5S rRNA is transcribed by RNA polymerase III separately (see reviews in Refs. 1-4). In yeast, 5S, 5.8S, and 25S (28S in higher eukaryotes) rRNA assemble with 39 ribosomal proteins to make up the 60S subunit, whereas 18S rRNA and 33 ribosomal proteins constitute the 40S subunit (5-7).Ribosome biogenesis is necessary for cell growth and proliferation. The synthesis of ribosome is a complex pathway in which over 200 trans-acting factors are involved. Many of the RNA folding, protein assembly, and maturation events are hierarchical, requiring the correct completion of one event to progress to the next event. This is seen in the assembly of ribosomal proteins onto the RNA in bacterial ribosome assembly (8) as well as in cytoplasmic maturation of the large subunit in yeast (9, 10). In addition, trans-acting factors also perform quality control steps for the assembly of the protein synthesis machinery. Syo1 facilitates the synchronized import of the two 5S rRNA-binding proteins, Rpl5 and Rpl11, to ensure stoichiometric the incorporation of these two proteins into the pre-60S subunits and also works as an assembly platform for the 5S ribonucleoprotein (RNP) (11,12). Release of the anti-association factor Tif6 requires elongation factor-like Efl1 and tRNAlike Sdo1 to confirm the integrity of the P-site (13-15). Transacting factors on cytoplasmic pre-40S subuni...

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“…After export from the nucleus, the release of Tif6 from the pre-60S subunit occurs via joint action of the GTPase Efl1 and Sdo1 (yeast orthologue of the SBDS protein) [15]. Sdo1/SBDS functions as the guanine exchange factor for EFL1 [16,17] that, upon GTP hydrolysis, displaces Tif6 by competing for a partial overlapping site on the surface of the 60S ribosomal subunit [18]. This last step promotes joining of the mature 60S ribosomal subunits with the 40S subunits to produce translationally competent ribosomes.…”

Section: Introductionmentioning

confidence: 99%

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

et al. 2018

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Abstract:The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome) protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N-and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

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Guanine nucleotide exchange in the ribosomal GTPase EFL1 is modulated by the protein mutated in the Shwachman–Diamond Syndrome

Cited by 23 publications

References 37 publications

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein

The Roles of Puf6 and Loc1 in 60S Biogenesis Are Interdependent, and Both Are Required for Efficient Accommodation of Rpl43

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

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