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Mapping protein interactions in the active TOM-TIM23 supercomplex

ABSTRACT: Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organisation of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modelling allowed us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF-X, Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Olexandr Dybkov

LAB HEAD: Prof. Dr. Henning Urlaub

PROVIDER: PXD028002 | Pride | 2021-12-13

REPOSITORIES: Pride

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Project description:Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and rRNA remodeling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors (AFs), organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these AFs function are largely unknown. Here, we use cryo-electron microscopy (cryo-EM), to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged AF Nog2. Our data pinpoint the locations and determine the structures of over 20 AFs, which are enriched in two areas, an arc region extending from the central protuberance (CP) to the polypeptide tunnel exit (PTE), and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S rRNAs. These structural data suggest that the arc-located factors might function to chaperone formation of RNA helices found in the active sites of the subunit, including helices from the CP, peptidyl-transferase center (PTC) and intersubunit bridge. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant AFs and functional rRNA elements, manifesting their critical roles in structural remodeling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodeling events before nuclear export: rotation of the 5S RNP, construction of the active center, and ITS2 removal. Therefore, our structures provide a framework to understand the molecular roles of diverse AFs, and potentially the atomic information therein constitutes a resource to generalize principles governing the elusive functions of nuclear RNA-binding proteins.

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Mapping protein interactions in the active TOM-TIM23 supercomplex

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