Biology subjects

Rapoport, T. A.

Publications and source records attributed to Rapoport, T. A..

9 recordsLinked to original sources

bioRxiv2026

Activation and inactivation pathways of a p53-like transcription factor govern lipid homeostasis in yeast

Membrane fluidity depends on unsaturated acyl chains that are generated in Saccharomyces cerevisiae by the desaturase Ole1, whose expression is primarily under the control of the transcription factor Mga2. Here, we show that the endoplasmic reticulum-anchored Mga2 precursor is ubiquitinated by the E3 ligase Rsp5 and then processively degraded by the proteasome until proteolysis stalls at a defined site, releasing a soluble fragment that enters the nucleus and activates Ole1 transcription. Unexpectedly, Mga2 contains a DNA-binding domain and a trans-activation-like segment structurally and functionally related to those of the human tumor suppressor p53. The mature transcription factor is degraded in the nucleus in a DNA binding-dependent manner; blocking this degradation causes unsaturated acyl chains to accumulate in lipid droplets, a detoxification response required for cell viability. These findings define the pathways that activate and inactivate Mga2, and uncover an evolutionary connection between the yeast lipid homeostasis regulator Mga2 and p53.

cell biology↗

bioRxiv2025

Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation

Misfolded or unassembled membrane proteins in the endoplasmic reticulum (ER) are polyubiquitinated, translocated into the cytosol, and degraded by the proteasome, a poorly understood process that is conserved in all eukaryotes. Here, we use S. cerevisiae to elucidate how ER membrane proteins are selected for degradation. We show that hydrophilic residues in a trans-membrane (TM) segment cause the TM to partition into a thinned membrane region next to the ubiquitin ligase Hrd1, which then leads to substrate polyubiquitination and degradation. In the case of single-pass membrane proteins, the Hrd1-associated Der1 protein contributes to partitioning and degradation. In contrast, multi-pass proteins require Hrd1 to function on its own. Our results provide a general mechanism by which ER membrane proteins are targeted for degradation.

cell biology↗

bioRxiv2023

Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation

Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex, rather than substrate recruitment. In vivo experiments confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.

biochemistry↗

bioRxiv2023

The membrane curvature inducing REEP1 proteins generate a novel ER-derived vesicular compartment

The endoplasmic reticulum (ER) is shaped by abundant, membrane curvature-generating proteins that include the REEP family member REEP5. The REEP1 subfamily, consisting of REEP1-4 in mammals, differs in abundance and topology from REEP5. Mutations in REEP1 and REEP2 cause Hereditary Spastic Paraplegia, but REEP1-4s function remains enigmatic. Here we show that the REEP1 proteins reside in a novel vesicular compartment and identify features that determine their localization. Mutations in REEP1 proteins that compromise curvature-inducing activity, including those that cause disease, relocalize the proteins to the bulk ER. These mutants interact with wildtype proteins to retain them in the ER, consistent with their autosomal-dominant disease inheritance. REEP1vesicles contain the fusogen atlastin-1, but not general ER proteins. We propose that REEP1 proteins generate these vesicles themselves by budding directly from the ER, and that they cycle back to the ER by atlastin-mediated fusion. The vesicles may serve to regulate ER tubule dynamics.

cell biology↗

bioRxiv2022

Membrane curvature-generating proteins crucial for autophagosome formation

Autophagy is essential for cellular homeostasis and begins with the formation of a phagophore, a cup-like membrane sheet consisting of two closely apposed lipid bilayers connected by a highly curved rim. How the membrane sheet forms, bends, and eventually generates an autophagosome that enwraps cargo remains enigmatic. Specifically, it is unclear how the high membrane curvature of the phagophore rim, an energetically unfavorable state, is stabilized. Here, we demonstrate that phagophore formation requires the conserved, membrane curvature-generating REEP1 proteins. The REEP1 family proteins (REEP1-4 in vertebrates) differ from the related endoplasmic reticulum-shaping REEPs in abundance and membrane topology. In fission yeast, the single REEP1 ortholog is involved in both bulk and selective autophagy. It is recruited at early stages of phagophore formation and required for their maturation into autophagosomes. The function of REEP1 relies on its ability to generate high membrane curvature and its localization to the phagophore rim. Mammalian REEP1 proteins also associate with phagophores upon induction of autophagy and colocalize with early autophagic markers. We propose that REEP1 proteins stabilize the phagophores highly curved rim so that the two membrane sheets are kept in close proximity to form the autophagosome. Defective autophagy may underlie the effect of curvature-compromising mutations in human REEP1 proteins linked to neurological disease.

cell biology↗

bioRxiv2022

Mechanism of PEX5-mediated protein import into peroxisomes

Peroxisomes are ubiquitous organelles, whose dysfunction causes fatal human diseases. Most peroxisomal enzymes are imported from the cytosol by the receptor PEX5, which interacts with a docking complex in the peroxisomal membrane, and then returns to the cytosol after monoubiquitination by a membrane-embedded ubiquitin ligase. The mechanism by which PEX5 shuttles between cytosol and peroxisomes, and releases cargo inside the lumen, is unclear. Here, we use Xenopus egg extract to demonstrate that PEX5 accompanies cargo completely into the lumen, utilizing WxxxF/Y motifs near its N-terminus that bind a lumenal domain of the docking complex. PEX5 recycling is initiated by an amphipathic helix that binds to the lumenal side of the ubiquitin ligase. The N-terminus then emerges in the cytosol for monoubiquitination. Finally, PEX5 is extracted from the lumen, resulting in unfolding of the receptor and cargo release. Our results reveal the unique mechanism by which PEX5 ferries proteins into peroxisomes.

cell biology↗

bioRxiv2021

Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

biochemistry↗

bioRxiv2021

Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: (1) a Fab-fragment of an antibody directed against the nanobody, and (2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of [~]120 kDa, called Legobody, does not perturb the nanobody-target interaction and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23 kDa membrane protein, resulting in a map at 3.2[A] overall resolution with density sufficient for de novo model building, and for the 22 kDa RBD of SARS-CoV2 spike protein, resulting in a map at 3.6 [A] resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

biochemistry↗

bioRxiv2017

Structure and function of accessory Sec proteins involved in the adhesin export pathway of Streptococcus gordonii

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. These adhesins are O-glycosylated by several cytosolic glycosyltransferases and require three accessory Sec proteins (Asp1-3) for export, but how the adhesins are processed for secretion is not well defined. Here, we show that O-glycosylation of S. gordonii adhesin GspB occurs in a sequential manner by three enzymes (GtfA/B, Nss, Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. The modified substrate is subsequently transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures show that both Asp1 and Asp3 are related to carbohydrate binding proteins. Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. These results suggest a mechanism for the modification of adhesin in the cytosol and its subsequent targeting to the export machinery.

biochemistry↗