A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast.

Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin-ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin-induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution.

Actively maintained close appositions between organelle membranes, also known as contact sites, enable the efficient transfer of biomolecules between cellular compartments. Several such sites have been described as well as their tethering machineries. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize contact site proteomes, we established a high-throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, several of which are poorly characterized, on the background of 1165 strains expressing a mCherry-tagged yeast protein that has a cellular punctate distribution (a hallmark of contact sites), under regulation of the strong TEF2 promoter. By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified three homologs of Vps13 and Atg2 that are residents of multiple contact sites. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell. Overall, our analysis expands the universe of contact site residents and effectors and creates a rich database to mine for new functions, tethers, and regulators.

A conserved viral amphipathic helix governs the replication site-specific membrane association.

Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins recognize and what motifs or domains in viral replication proteins determine their destinations. We show here that an amphipathic helix, helix B in replication protein 1a of brome mosaic virus (BMV), is necessary for 1a's localization to the nuclear endoplasmic reticulum (ER) membrane where BMV assembles its VRCs. Helix B is also sufficient to target soluble proteins to the nuclear ER membrane in yeast and plant cells. We further show that an equivalent helix in several plant- and human-infecting viruses of the Alsuviricetes class targets fluorescent proteins to the organelle membranes where they form their VRCs, including ER, vacuole, and Golgi membranes. Our work reveals a conserved helix that governs the localization of VRCs among a group of viruses and points to a possible target for developing broad-spectrum antiviral strategies.

Cnm1 mediates nucleus-mitochondria contact site formation in response to phospholipid levels.

Mitochondrial functions are tightly regulated by nuclear activity, requiring extensive communication between these organelles. One way by which organelles can communicate is through contact sites, areas of close apposition held together by tethering molecules. While many contacts have been characterized in yeast, the contact between the nucleus and mitochondria was not previously identified. Using fluorescence and electron microscopy in S. cerevisiae, we demonstrate specific areas of contact between the two organelles. Using a high-throughput screen, we uncover a role for the uncharacterized protein Ybr063c, which we have named Cnm1 (contact nucleus mitochondria 1), as a molecular tether on the nuclear membrane. We show that Cnm1 mediates contact by interacting with Tom70 on mitochondria. Moreover, Cnm1 abundance is regulated by phosphatidylcholine, enabling the coupling of phospholipid homeostasis with contact extent. The discovery of a molecular mechanism that allows mitochondrial crosstalk with the nucleus sets the ground for better understanding of mitochondrial functions in health and disease.

Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling.

Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.

Double the Fun, Double the Trouble: Paralogs and Homologs Functioning in the Endoplasmic Reticulum.

The evolution of eukaryotic genomes has been propelled by a series of gene duplication events, leading to an expansion in new functions and pathways. While duplicate genes may retain some functional redundancy, it is clear that to survive selection they cannot simply serve as a backup but rather must acquire distinct functions required for cellular processes to work accurately and efficiently. Understanding these differences and characterizing gene-specific functions is complex. Here we explore different gene pairs and families within the context of the endoplasmic reticulum (ER), the main cellular hub of lipid biosynthesis and the entry site for the secretory pathway. Focusing on each of the ER functions, we highlight specificities of related proteins and the capabilities conferred to cells through their conservation. More generally, these examples suggest why related genes have been maintained by evolutionary forces and provide a conceptual framework to experimentally determine why they have survived selection.

Germinality does not necessarily define mAb expression and thermal stability.

The production potential of recombinant monoclonal antibody (mAb) expressing cell lines depends, among other factors, on the intrinsic antibody structure determined by the amino acid sequence. In this study, we investigated the influence of somatic mutations in the V(D)J sequence of four individual, mature model mAbs on the expression potential. Therefore, we defined four couples, each consisting of one naturally occurring mAb (2G12, Ustekinumab, 4B3, and 2F5) and the corresponding germline-derived cognate mAb (353/11, 554/12, 136/63, and 236/14). For all eight mAb variants, recombinant Chinese hamster ovary (CHO) cell lines were developed with mAbs expressed from a defined chromosomal locus. The presented workflow investigates critical parameters including productivity, intra- and extracellular product profile, XBP1 splicing, thermal stability, and in silico hydrophobicity. Significant differences in productivity were even observed between the germline-derived mAbs which did not undergo somatic mutagenesis. Accordingly, back-to-germline mutations of mature mAbs are not necessarily reflecting improved expression and stability but indicate opportunities and limits of mAb engineering. From our studies, we conclude that germinalization represents a potential to improve mAb properties depending on the antibody's germline family, highlighting the fact that mAbs should be treated individually.

An essential role for the Zn2+ transporter ZIP7 in B cell development.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

ERAD-dependent control of the Wnt secretory factor Evi.

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of ß-catenin by the ubiquitin-proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)-associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP-dependent manner. Ubiquitination of Evi involves the E2-conjugating enzyme UBE2J2 and the E3-ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD-dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.

Conserved cytoplasmic domains promote Hrd1 ubiquitin ligase complex formation for ER-associated degradation (ERAD).

The mammalian ubiquitin ligase Hrd1 is the central component of a complex facilitating degradation of misfolded proteins during the ubiquitin-proteasome-dependent process of ER-associated degradation (ERAD). Hrd1 associates with cofactors to execute ERAD, but their roles and how they assemble with Hrd1 are not well understood. Here, we identify crucial cofactor interaction domains within Hrd1 and report a previously unrecognised evolutionarily conserved segment within the intrinsically disordered cytoplasmic domain of Hrd1 (termed the HAF-H domain), which engages complementary segments in the cofactors FAM8A1 and Herp (also known as HERPUD1). This domain is required by Hrd1 to interact with both FAM8A1 and Herp, as well as to assemble higher-order Hrd1 complexes. FAM8A1 enhances binding of Herp to Hrd1, an interaction that is required for ERAD. Our findings support a model of Hrd1 complex formation, where the Hrd1 cytoplasmic domain and FAM8A1 have a central role in the assembly and activity of this ERAD machinery.

New MAPS for misfolded proteins.

Clearing misfolded proteins from the cytoplasm is essential to maintain cellular homeostasis. Now, a parallel clearance system is described that uses the deubiquitylase USP19 to enable secretion of misfolded cytoplasmic proteins when conventional proteasomal degradation is compromised. Misfolding-associated protein secretion (MAPS) has important implications for protein quality control and prion-like transmission.

The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins.

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.