Proteomics

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LC-MS/MS analysis of ribosome UFMylation in vitro

ABSTRACT: UFMylation, is a Ubiquitin-like modification occurring on RPL26, a ribosomal subunit, upon ribosome stalling at the ER membrane. This modification is achieved by a pool of enzymes commonly referred to as E1-activating, E2-conjugating and E3-ligase enzymes. The UFM1 E3 ligase comprises of three proteins: UFL1, UFBP1 and CDK5RAP3, referred to as UFM1 Ribosome E3 Ligase (UREL) complex. While UFL1 and UFBP1 are sufficient for E3 ligase activity in vitro, CDK5RAP3 is indispensable for ribosome UFMylation in vivo. In this work, we characterize in vitro UFMylation reaction of purified 60S ribosomes in the presence and absence of CDK5RAP3 using LC-MS/MS. Specifically, we analyse sites of UFMylation, linkage type and quantify monoUFMylation and diUFMylation on RPL26. Interestingly, we find UFMylation to occur on a specific lysine residue, K134, in the presence of UFL1/UFBP1 alone and in complex with CDK5RAP3. In addition, we also provide quantitative information on relative abundances of RPL26 mono- and di-UFMylation under these conditions.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Frederic Lamoliatte  

LAB HEAD: Yogesh Kulathu

PROVIDER: PXD046991 | Pride | 2024-03-22

REPOSITORIES: Pride

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Publications

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)<sup>1,2</sup>. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. <sup>3</sup>). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscop  ...[more]

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