Biology subjects

Pillet, B.

Publications and source records attributed to Pillet, B..

3 recordsLinked to original sources

bioRxiv2025

Exploration of the proxiOME of large subunit ribosomal proteins reveals Acl1 and Bcl1 as cooperating dedicated chaperones of Rpl1

In eukaryotes, most newly synthesized ribosomal proteins (r-proteins) need to rapidly and safely get into the nucleus to reach their assembly site on pre-ribosomal particles. However, only for few r-proteins tailored support mechanisms involving so-called dedicated chaperones could so far be revealed. Here, with the primary aim of identifying novel dedicated chaperones, we performed TurboID-based proximity labelling with all 46 large subunit r-proteins of Saccharomyces cerevisiae, which unveiled the fungi-specific Acl1 and the conserved Bcl1 as candidate dedicated chaperones of Rpl1. We show that the functionally cooperating Acl1 and Bcl1 both directly interact with Rpl1, form a trimeric Acl1-Rpl1-Bcl1 complex, and enable the nuclear import of Rpl1. Moreover, our crystal structure of the minimal Acl1-Rpl1 complex reveals how Acl1s ankyrin repeat domain shields a positively charged rRNA-binding surface of Rpl1. Our proximity labelling approach also permitted to establish novel interactions between four r-proteins and distinct importins and to illuminate r-protein neighbourhoods on successive pre-60S particles. Additionally, reciprocal proximity labelling with the known dedicated chaperones indicates that almost all appear to be transiently associated with pre-ribosomal particles. Our study provides for the first time comprehensive insight into the physical proximities of large subunit r-proteins along their entire life cycle. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/677003v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1167c6corg.highwire.dtl.DTLVardef@1429c23org.highwire.dtl.DTLVardef@18f7ecorg.highwire.dtl.DTLVardef@13ccdfb_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

biochemistry↗

bioRxiv2023

The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization

Canonical autophagy is regulated by ULK1, the most proximal protein kinase specifically regulating autophagy initiation. To gain new insights into functions of the ULK1 holo-complex in autophagy regulation, we generated a deep ULK1 complex interactome by combining affinity purification- and proximity labelling-mass spectrometry of all four ULK1 complex members: ULK1, ATG13, ATG101 and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases implying the formation of a signalosome. Interestingly, also several selective autophagy receptors interact with ULK1 indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth promoting conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, supporting its recruitment to the ER membrane and positively affecting autophagy flux.

cell biology↗

bioRxiv2021

Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis

The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, prevents the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

cell biology↗