Search

Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells.

Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum-associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study, we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.

Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.

Author(s): Eric J. Bennett [1]; Thomas A. Shaler [2]; Ben Woodman [3]; Kwon-Yul Ryu [1]; Tatiana S. Zaitseva [1]; Christopher H. Becker [2]; Gillian P. Bates [3]; Howard Schulman [2]; [...]

I. INTRODUCTION The autosomal recessive human genetic disorder cystic fibrosis (CF) is caused by the loss or dysfunction of a plasma membrane [C1.sup.-] channel known as the CF transmembrane conductance [...]

The effects of morphological changes in the cystic fibrosis transmembrane regulator chloride (CFTR GCl-) conductance was examined in the apical membranes of the sweat ducts. The luminal pH and cytoplasmic pH were specifically analyzed. The reciprocal control of relative kinase phosphorylation and phosphatase dephosphorylation contributed to CFTR GCl- modulation. Moreover, CFTR GCl was independent of extracytosolic luminal pH in the range of 5.0-8.5.

The endoplasmic reticulum (ER) performs several functions. It is the port of entry of membrane and secretory proteins into the central vacuolar system. It also serves as the site of maturation of nascent secretory and membrane proteins to their respective tertiary and quaternary structures. The ER is equipped with a 'quality control' mechanism through which proteins that fail to fold or oligomerize correctly are not allowed to be secreted and are degraded. Such process appears to occur within the organelle.

A study on the regulatory mechanism of the nonerythroid anion exchanger AE2 by the intracellular protons, reveals its high sensitivity to intracellular pH. The intracellular pH sensor is determined to be the four histidine residues inside the N-terminal, cytoplasmic domain of the pH-sensitive AE2 and AE3 exchangers. This domain is not found in the erythroid anion exchanger. The determined pH sensitivity AE2 and AE3 are consistent with their physiological activities. A new feedback mechanism is also proposed involving direct control of anion exchangers by intracellular protons.

Rapid cystic fibrosis transmembrane conductance regulator (CFTR) degradation occurs due to ubiquitination. Two strong proteasome inhibitors induce aggregation of polyubiquitinated forms of immature CFTR and inhibit degradation of wild-type and mutant CFTR. Coexpression of a dominant negative ubiquitin mutant also inhibits degradation of CFTR. Degradation is also suppressed in cells having a temperature-sensitive mutation in ubiquitin-activating enzyme.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding channel (ABC) transporter and a plasma membrane chloride channel. CFTR is of the traffic ATPase superfamily and chlorine ion channel that seems to need ATP hydrolysis for gating. Investigation of single CFTR chlorine ion channels showed the existence of two open conductance states that are related to each other and to the closed state by an asymmetric cycle of gating events.