Structural studies of eukaryotic ribosome biogenesis and the sec and Bcs1 protein translocation systems

Kater, Lukas Jonas (2020): Structural studies of eukaryotic ribosome biogenesis and the sec and Bcs1 protein translocation systems. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Three publications of this cumulative dissertation use cryo-electron microscopy (cryo-EM) to dissect the assembly pathway of the eukaryotic large ribosomal subunit (LSU). This pathway commences with freshly transcribed and initially unfolded rRNA in the nucleolus, which folds and incorporates ribosomal proteins while traveling to the cytoplasm, ultimately culminating in the mature LSU. During this highly complex pathway, the yeast cell must assemble four rRNAs and 79 ribosomal proteins with the help of over 200 assembly factors (AFs). Using cryo-EM, structures of nucleo\-plasmic and cytoplasmic assembly intermediates of the LSU could be solved in recent years, thus shedding light on the later stages of LSU formation. Early assembly steps remain enigmatic, as nucleolar LSU assembly intermediates have been biochemically but not structurally characterized. Taken together, we solved the structure of seven nucleolar or early nucleoplasmic intermediates at resolutions ranging from 3.3 to 4.5 Å, showing a linear assembly sequence. The first five structures show how the rRNA of the LSU is incorporated stepwise, in a non-transcriptional sequence, first forming the solvent exposed back side, and later the peptide exit tunnel and parts of the intersubunit surface (ISS). At the late nucleolar stage, the L1-stalk rRNA of domain V blocks the site of central protuberance (CP) assembly and is stabilized in a premature conformation by a range of AFs associated with the meandering, long N-terminal tail of Erb1. Two further structures show progression from this stage after release of the Erb1-Ytm1 complex by the Rea1 remodeling machinery. These intermediates, purified via Nop53, show dissociation of many early AFs from the premature ISS and destabilization of the L1-stalk. After subsequent release of the Spb1 methyltransferase, the L1-stalk rRNA can be accommodated in its mature conformation. This allows the premature CP to form, leading to a previously characterized nucleoplasmic intermediate, with a formed but premature CP. This particle is the substrate for the second Rea1 mediated structural remodeling, an intermediate of which we resolved to molecular resolution revealing Ipi1 as a central integrator for the Rix1-Ipi1-Ipi3 complex on this pre-60S particle. The binding of the Rix1-Rea1 remodeling machinery at this nucleoplasmic stage progresses maturation by inducing a 180$^{\circ}$ rotation of the 5S ribonucleoprotein particle (5S RNP) and CP. Using a combination of yeast genetics and cryo-EM we investigated the function of the AF Cgr1 in this maturation step. We showed that Cgr1 is required for CP rotation to take place, likely by stabilizing the rotated conformation. The Cgr1 function can be bypassed by introducing suppressor mutations in Rpf2 and Rrs1, two factors stabilizing the CP prior to rotation. Apart from ribosome biogenesis, two additional publications of this dissertation address protein translocation machinery, required for transport of proteins across or into membranes. The Sec translocon allows co- and posttranslational translocation of mostly unfolded substrates across the bacterial plasma and the eukaryotic endoplasmic reticulum (ER) membrane. We solved the structure of a stalled 70S ribosome-nascent chain complex (RNC) bound to the SecYEG translocon in a native like environment provided by a large lipid nanodisc. The structure shows all three subunits of the bacterial SecYEG complex and displays the lateral gate at a defined, early stage of opening or unzipping on the cytoplasmic side upon insertion of the signal anchor domain of the nascent chain. Specific pathways, such as the assembly of the mitochondrial bc1 respiratory chain complex, require folding of proteins in one compartment before translocation across a membrane to allow the protein to be active in another compartment. The bc1-complex component Rip1 folds in the mitochondrial matrix and assembles a 2Fe-2S cluster before being translocated across the inner mitochondrial membrane (IM) by the AAA-protein Bcs1. We solved the structure of Bcs1 in an ADP-bound state and two apo states, displaying a heptameric ring of Bcs1 protomers. Bcs1 forms two large aqueous vestibules separated by a seal forming middle domain. One vestibule is accessible from the matrix side and one lies within the inner mitochondrial membrane. The architecture and structural dynamics between the three states suggest an airlock like mechanism, allowing transport of folded Rip1 while maintaining the permeability barrier of the membrane.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 10. Juli 2020
• 1. Bericht­erstatter:in: Beckmann, Roland