Loss of Grp170 results in catastrophic disruption of endoplasmic reticulum function.

GRP170 (Hyou1) is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds nonnative proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of mouse embryonic fibroblasts obtained from mice in which LoxP sites were engineered in the Hyou1 loci (Hyou1LoxP/LoxP). A doxycycline-regulated Cre recombinase was stably introduced into these cells. Induction of Cre resulted in depletion of Grp170 protein which culminated in cell death. As Grp170 levels fell we observed a portion of BiP fractionating with insoluble material, increased binding of BiP to a client with a concomitant reduction in its turnover, and reduced solubility of an aggregation-prone BiP substrate. Consistent with disrupted BiP functions, we observed reactivation of BiP and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and provide hypotheses as to why mutations in the Hyou1 locus are linked to human disease.

Loss of Grp170 results in catastrophic disruption of endoplasmic reticulum functions.

GRP170, a product of the Hyou1 gene, is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds non-native proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of embryonic fibroblasts from mice in which LoxP sites were engineered in the Hyou1 loci ( Hyou1 LoxP/LoxP ). A doxycycline-regulated Cre recombinase was also stably introduced into these cells. Induction of Cre resulted in excision of Hyou1 and depletion of Grp170 protein, culminating in apoptotic cell death. As Grp170 levels fell we observed increased steady-state binding of BiP to a client, slowed degradation of a misfolded BiP substrate, and BiP accumulation in NP40-insoluble fractions. Consistent with disrupted BiP functions, we observed reactivation of BiP storage pools and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and insights into mutations in the Hyou1 locus and human disease.

Identification of two rate-limiting steps in the degradation of partially folded immunoglobulin light chains.

Antibody monomers are produced from two immunoglobulin heavy chains and two light chains that are folded and assembled in the endoplasmic reticulum This process is assisted and monitored by components of the endoplasmic reticulum quality control machinery; an outcome made more fraught by the unusual genetic machinations employed to produce a seemingly unlimited antibody repertoire. Proper functioning of the adaptive immune system is as dependent on the success of this operation, as it is on the ability to identify and degrade those molecules that fail to reach their native state. In this study, two rate-limiting steps were identified in the degradation of a non-secreted κ light chain. Both focus on the constant domain (CL), which has evolved to fold rapidly and very stably to serve as a catalyst for the folding of the heavy chain CH1 domain. The first hurdle is the reduction of the disulfide bond in the CL domain, which is required for retrotranslocation to the cytosol. In spite of being reduced, the CL domain retains structure, giving rise to the second rate-limiting step, the unfolding of this domain at the proteasome, which results in a stalled degradation intermediate.