Biology subjects

Page, K. R.

Publications and source records attributed to Page, K. R..

4 recordsLinked to original sources

bioRxiv2026

TXNDC15 modulated quality control at the endoplasmic reticulum shapes ciliogenesis

At the endoplasmic reticulum (ER), membrane protein quality control is tightly regulated to ensure excess subunits are recognized and degraded to protect cellular homeostasis. Using genome wide CRISPR screens, we identified a factor of unknown function, thioredoxin domain containing protein 15 (TXNDC15), and showed that it regulates membrane protein stability by tuning the activity of the E3-ubiquitin ligase, MARCHF6. TXNDC15 modulates MARCHF6 in two opposing ways: first, it enhances the binding, ubiquitination, and degradation of membrane protein subunits with soluble cytosolic domains; and second, it prevents the inappropriate recruitment and ubiquitination of subunits with globular lumenal domains. Patient mutations to TXNDC15 that cause the ciliopathy Meckel-Gruber syndrome, disrupted its binding to MARCHF6, allowing degradation of critical ciliary proteins as they transit through the ER leading to defects in ciliogenesis. The regulatory function of TXNDC15 therefore exemplifies how protein quality control maintains the integrity of the proteome to prevent disease.

molecular biology↗

bioRxiv2023

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC*BOS holocomplex showed that characteristics of a GPCRs soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, multipass translocon, and Sec61 for biogenesis of diverse membrane proteins in human cells.

cell biology↗

bioRxiv2023

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches.

genomics↗

bioRxiv2019

Differential modes of orphan subunit recognition for the WRB/CAML complex

A large proportion of membrane proteins must be assembled into oligomeric complexes for function. How this process occurs is poorly understood, but it is clear that complex assembly must be tightly regulated to avoid accumulation of orphan subunits with potential cytotoxic effects. We interrogated assembly in mammalian cells using a model system of the WRB/CAML complex: an essential insertase for tail-anchored proteins in the endoplasmic reticulum (ER). Our data suggests that the stability of each subunit is differentially regulated. In WRBs absence, CAML folds incorrectly, causing aberrant exposure of a hydrophobic transmembrane domain to the cytosol. When present, WRB can post-translationally correct the topology of CAML both in vitro and in cells. In contrast, WRB can independently fold correctly, but is still degraded in the absence of CAML. We therefore propose at least two distinct regulatory pathways for the surveillance of orphan subunits during complex assembly in the mammalian ER.

molecular biology↗