Biology subjects

Lederkremer, G. Z.

Publications and source records attributed to Lederkremer, G. Z..

5 recordsLinked to original sources

bioRxiv2026

Mannosidases IA, IB and IC are in segregated vesicular structures and involved in both glycoprotein quality control and maturation

N-linked glycoprotein processing critically depends on the trimming of -1,2 mannose residues, a key step required both for glycoprotein maturation along the secretory pathway and for targeting defective glycoproteins to endoplasmic reticulum-associated degradation (ERAD). Mammalian cells express seven Class I -1,2 mannosidases, yet their individual roles remain poorly defined, particularly for ManIA, ManIB, and ManIC, which were originally considered Golgi-resident maturation enzymes. Here, we re-evaluated the subcellular localization and functional contributions of these three mannosidases to glycoprotein quality control and maturation. We found that ManIA, ManIB, and ManIC localize predominantly to quality control vesicles (QCVs), previously identified by our group, whereas only ManIC displays a substantial Golgi population. Surprisingly, each enzyme is confined to a different vesicular population. All three enzymes promote ERAD targeting of misfolded model glycoproteins, albeit with different substrate preferences. In addition, they redundantly support the maturation and cell-surface delivery of a model glycoprotein. Most strikingly, in vitro analyses revealed that ManIA, ManIB, and ManIC preferentially trim a properly folded model glycoprotein rather than its denatured form. This is the opposite of the substrate preference that we previously observed for ERManI, EDEM1, and EDEM2. These findings support a model in which ERManI and the EDEMs selectively process misfolded glycoproteins to promote their recognition by the proximal lectin OS-9 and subsequent ERAD. In contrast, ManIA, ManIB and ManIC can slowly process misfolded glycoproteins but act rapidly on properly folded glycoprotein molecules, releasing them from ER-Golgi lectin-mediated retention or retrieval pathways, thereby promoting forward trafficking and maturation in the Golgi.

cell biology↗

bioRxiv2026

ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD), a process requiring their exit from the ER and delivery to cytosolic proteasomes via the key retrotranslocation step. Conventional assays detect fully extracted substrates, overlooking early initiation events. Here, we use an Escherichia coli-derived Biotin Acceptor Peptide (BAP)-biotin ligase BirA system to detect ERAD substrates upon cytosolic exposure. Using the model ERAD substrate H2a, we show that cytosolic BirA selectively biotinylates luminal BAP tags during early cytosolic exposure, enabling detection of substrates engaging the mammalian retrotranslocation machinery independently of subsequent extraction. Combined with high-resolution expansion fluorescence microscopy, this approach reveals active initiation of ERAD at the specialized ER-derived quality control compartment (ERQC). Substrates become transiently exposed to the cytosol while remaining membrane-associated, followed by tight coupling of polyubiquitination, transmembrane segment extraction, deglycosylation and degradation. Functional perturbations indicate that mannose trimming enables initiation, whereas p97 and HRD1 support later extraction and ubiquitination.

cell biology↗

bioRxiv2025

Helical charge distribution at the transmembrane-luminal interface determines subcellular localization

Transmembrane protein localization is typically dictated by short cytosolic tail sequences, whereas luminal domain contribution remains less understood. Here, we identify a short luminal juxtamembrane peptide as a key determinant of subcellular localization for Class I -1,2 mannosidases--ERManI, ManIA, ManIB, and ManIC--members of the glycoside hydrolase 47 family involved in N-glycoprotein processing. We previously showed that ERManI and ManIA localize to specialized quality control vesicles (QCVs); we now find that ManIB also partially localizes to these vesicles, whereas ManIC is predominantly Golgi-localized. ERManI, ManIA, and ManIB share a conserved luminal juxtamembrane distribution of charged residues, which diverges in ManIC. Structural predictions suggest that this region maintains an -helical conformation, with the charge pattern oriented on one face. Site-directed mutagenesis that disrupted this charge pattern or altered its helical register (via alanine insertions) shifted localization between QCVs and the Golgi. Moreover, grafting this peptide onto an unrelated transmembrane protein, {beta}-1,3-galactosyltransferase, redirected it from the Golgi to QCVs. These findings suggest that a specific three-dimensional charge pattern at the transmembrane-luminal interface serves as a localization signal. Unlike canonical linear motifs, this mechanism relies on the structural arrangement, revealing a previously unrecognized mode of organelle targeting within the secretory pathway.

cell biology↗

bioRxiv2024

Oligosaccharyltransferase is involved in targeting to ER-associated degradation

Most membrane and secretory proteins undergo N-glycosylation, catalyzed by oligosaccharyltransferase (OST), a membrane-bound complex in the endoplasmic reticulum (ER). Proteins failing quality control are degraded via ER-associated degradation (ERAD), involving retrotranslocation to cytosolic proteasomes. Using SILAC proteomics, we identified OST subunits as key interactors with a misfolded ER protein bait, suggesting unexpected involvement in ERAD. Previous reports implied additional roles for OST subunits beyond N-glycosylation, such as quality control by ribophorin I. We tested OST engagement in glycoprotein and non-glycosylated protein ERAD; overexpression or partial knockdown of OST subunits interfered with ERAD in conditions that did not affect glycosylation. Effects were studied on misfolded membrane proteins, BACE476 and asialoglycoprotein receptor H2a, and the luminal 1-antitrypsin NHK variant. OST appears to participate in late ERAD stages, interacting with the E3 ligase HRD1 and facilitating retrotranslocation. Molecular dynamics simulations suggest membrane thinning by OST transmembrane domains, possibly assisting retrotranslocation via membrane distortion.

cell biology↗

bioRxiv2022

COP I and II dependent trafficking controls ER-associated degradation

Misfolded proteins and components of the endoplasmic reticulum (ER) quality control and ER associated degradation (ERAD) machineries concentrate in mammalian cells in the pericentriolar ER-derived quality control compartment (ERQC), suggesting it as a staging ground for ERAD. By tracking the chaperone calreticulin and an established ERAD substrate, asialoglycoprotein receptor H2a, we have now determined that the trafficking to the ERQC is reversible and recycling back to the ER is slower than the movement in the ER periphery. The dynamics suggest vesicular trafficking rather than diffusion. Indeed, using dominant negative mutants of ARF1 and Sar1 or the drugs Brefeldin A and H89, we observed that COPI inhibition causes accumulation in the ERQC and increased retrotranslocation and ERAD, whereas COPII inhibition has the opposite effect. Our results suggest that targeting of misfolded proteins to ERAD involves COPII-dependent transport to the ERQC and that they can be retrieved to the peripheral ER in a COPI-dependent manner.

cell biology↗