Identification of Neighboring Protein Pairs Cross-Linked with Dimethyl 3,3′-Dithiobispropionimidate in Rat Liver 40S Ribosomal Subunits1

Uchiumi, Toshio; Terao, Kazuo; Ogata, Kazuhiro

doi:10.1093/oxfordjournals.jbchem.a133449

Cited by 25 publications

(12 citation statements)

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“…However, crosslinking studies with 40S subunits have identiWed crosslinks of Rps19p Fig. 1 Positions of the mTn3-lacZ/LEU2 insertion sites in the isolated nep1-1 ts suppressors with Rps18p, Rps16p, Rps5p, and other proteins (Terao et al 1980;Tolan and Traut 1981;Uchiumi et al 1981;Yeh et al 1986). Based on cryo-EM data, the yeast 40S subunit structure could be modeled (Spahn et al 2001).…”

Section: Localization Of the Rps19p 18s Rrna Binding Sitementioning

confidence: 99%

“…4 Topography of Rps19p in the Saccharomyces cerevisiae 40S ribosome subunit. Predicted localization of Rps19p in the S. cerevisiae 40S ribosome subunit on the basis of protein-protein crosslink data (adapted from Terao et al 1980;Uchiumi et al 1981;Tolan and Traut 1981;Yeh et al 1986). The 40S cryo-EM structure model was modiWed from Spahn et al (2001) in contrast to the previously described multicopy suppressor SAM2, encoding S-adenosylmethionine synthetase 2, which only suppressed the nep1-1 ts mutation (Eschrich et al 2002).…”

Section: Suppressors Snr57 and Multicopy-rps19b Act Additivelymentioning

confidence: 99%

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Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

et al. 2006

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The nucleolar protein Nep1 and its human homologue were previously shown to be involved in the maturation of 18S rRNA and to interfere directly or indirectly with a methylation reaction. Here, we report that the loss-of-function mutation Deltasnr57 and multicopy expression of the ribosomal 40S subunit protein 19 (Rps19p) can partially suppress the Saccharomyces cerevisiae Deltanep1 growth defect. SnR57 mediates 2'-O-ribose-methylation of G(1570) in the 18S rRNA. By performing a three-hybrid screen, we isolated several short RNA sequences with strong binding affinity to Nep1p. All isolated RNAs shared a six-nucleotide consensus motif C/UUCAAC. Furthermore, one of the isolated RNAs exactly corresponded to nucleotides 1553-1577 of the 18S rRNA, which includes G(1570), the site of snR57-dependent 18S rRNA methylation. From protein-protein crosslink data and the cryo-EM map of the S. cerevisiae small ribosomal subunit, we suggest that Rps19p is localized in close vicinity to the Nep1p 18S rRNA binding site. Our results suggest that Nep1p binds adjacent to helix 47 of the 18S rRNA and possibly supports the association of Rps19p to pre-ribosomal particles.

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Section: Localization Of the Rps19p 18s Rrna Binding Sitementioning

confidence: 99%

Section: Suppressors Snr57 and Multicopy-rps19b Act Additivelymentioning

confidence: 99%

Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

et al. 2006

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“…Thus, four dimers were described by Westermann et al (1980), eight by Terao et al (1980), 10 by Uchiumi et al (1981), and 36 by Tolan and Traut (1981 (Terao et al, 1980), dimethyl 3,3' -dithiobispropionimidate (Uchiumi et al, 1981), 2-iminothiolane (Tolan and Traut, 1981), and DBI (Westermann et al, 1980, and two different species were analysed: rat liver (Terao et al, 1980;Westermann et al, 1980;Uchiumi et al, 1981; this study) and rabbit reticulocytes (Tolan and Traut, 1981). Furthermore, certainly none of the methods used permits the identification of all cross-linked protein dimers for a complete comparison.…”

Section: Discussionmentioning

confidence: 98%

“…bDBI (Westermann et al, 1980 and this paper). cDimethyl 3,3'-dithiobispropionimidate (Uchiumi et al, 1981).…”

Section: Discussionmentioning

confidence: 99%

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Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking.

1983

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Cross-linking of proteins within the small subunit of rat liver ribosomes by the bifunctional reagent dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate produced numerous covalently linked protein dimers which could be separated by a combination of ion-exchange chromatography on carboxymethyl cellulose and polyacrylamide gel electrophoresis. The protein components of the dimers were identified electrophoretically after periodate cleavage of the cross-link(s). The analysis revealed 42 cross-linked dimers involving 25 different proteins. Among these, proteins S3, S4 and S20 occurred in combinations with six, eight and seven different proteins, respectively. For proteins S13, S14 and S17 five protein neighbours could be identified, while 13 of the remaining proteins were linked to three or four different protein partners. The involvement of the majority of proteins in the formation of multiple cross-linked dimers implies that a large number of protein-protein interaction sites exist within the ribosomal subunit. A preliminary model illustrating the arrangement of 16 proteins in the small ribosomal subunit is presented and discussed with respect to possible functions, especially in the event of translation initiation.

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Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes.

Nygård¹,

Nika²

1982

The EMBO Journal

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Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross‐linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA‐bound, labelled proteins were identified by two‐dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.

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Identification of Neighboring Protein Pairs Cross-Linked with Dimethyl 3,3′-Dithiobispropionimidate in Rat Liver 40S Ribosomal Subunits1

Cited by 25 publications

References 0 publications

Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking.

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes.

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