Biology subjects

Schulman, B.

Publications and source records attributed to Schulman, B..

5 recordsLinked to original sources

bioRxiv2022

In situ structural analysis reveals membrane shape transitions during autophagosome formation

Autophagosomes are unique organelles which form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves a series of membrane transformations with distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (ET) and novel data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. By mapping individual structures onto a timeline based on geometric features, we uncover dynamic changes in membrane shape and curvature. Moreover, we reveal the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles such as the vacuole and the ER. Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores towards closure without a templating cargo.

cell biology↗

bioRxiv2022

Differential UBE2H-CTLH E2-E3 ubiquitylation modules regulate erythroid maturation

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganisation and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin (UB) system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the UB system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed distinct maturation stage-dependent assemblies of structurally homologous RANBP9-and RANBP10-CTLH complexes. Moreover, protein abundance of CTLHs cognate E2-conjugating enzyme UBE2H increased during terminal differentiation, which depended on catalytically active CTLH E3 complexes. CRISPR-Cas9 mediated inactivation of all CTLH E3 assemblies by targeting the catalytic subunit MAEA, or UBE2H, triggered spontaneous and accelerated maturation of erythroid progenitor cells including increased heme and haemoglobin synthesis. Thus, the orderly progression of human erythropoiesis is controlled by the assembly of distinct UBE2H-CTLH modules functioning at different developmental stages.

cell biology↗

bioRxiv2021

A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters

Cells rapidly remodel their proteomes to align their cellular metabolism to environmental conditions. Ubiquitin E3 ligases enable this response, by facilitating rapid and reversible changes to protein stability, localization, or interaction partners. In S. cerevisiae, the GID E3 ligase regulates the switch from gluconeogenic to glycolytic conditions through induction and incorporation of the substrate receptor subunit Gid4, which promotes the degradation of gluconeogenic enzymes. Here, we show an alternative substrate receptor, Gid10, which is induced in response to changes in temperature, osmolarity and nutrient availability, and regulates the ART-Rsp5 pathway. Art2 levels are elevated upon GID10 deletion, a crystal structure shows the basis for Gid10-Art2 interactions, and Gid10 directs a GID E3 ligase complex to ubiquitinate Art2. We also find that the GID E3 ligase affects the flux of plasma membrane nutrient transporters during heat stress. The data reveal GID as a system of E3 ligases with metabolic regulatory functions outside of glycolysis and gluconeogenesis, controlled by distinct stress-specific substrate receptors.

cell biology↗

bioRxiv2020

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies revealed at least two separable EMC activities, as an insertase regulating tail-anchored protein levels and as a polytopic membrane protein holdase chaperone. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a novel, non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMCs multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

biophysics↗

bioRxiv2019

Quantifying the heterogeneity of macromolecular machines by mass photometry

Sample purity is central to in vitro studies of protein function and regulation, as well as to the efficiency and success of structural studies requiring crystallization or computational alignment of individual molecules. Here, we show that mass photometry (MP) accurately reports on sample heterogeneity using minimal volumes with molecular resolution within minutes. We benchmark our approach by negative stain electron microscopy (nsEM), including workflows involving chemical crosslinking and multi-step purification of a multi-subunit ubiquitin ligase. When applied to proteasome stability, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP for rapid sample characterization, prioritization and optimization.

biophysics↗