ABSTRACT
Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric, IP(3)-gated channels in endoplasmic reticulum membranes that govern the release of Ca(2+) from this organelle. In response to activation of certain G protein-coupled receptors that persistently elevate IP(3) concentration, IP(3) receptors are ubiquitinated and degraded by the ubiquitin-proteasome pathway. IP(3) receptor ubiquitination is mediated by the ubiquitin-conjugating enzyme, (mam)Ubc7, a component of the endoplasmic reticulum-associated degradation pathway. However, the mechanism by which ubiquitinated IP(3) receptors are transferred to the proteasome is not known. Here, we examine this process and show in several mammalian cell types that the ATPase p97 associates with IP(3) receptors in response to hormonal stimuli that induce IP(3) receptor ubiquitination. To examine the functional relevance of the p97 interaction with IP(3) receptors, we stably and specifically reduced p97 protein levels by 62 +/- 3% in Rat-1 fibroblasts using RNA interference. In these cells, endothelin-1-induced IP(3) receptor degradation was markedly retarded and the accumulation of ubiquitinated IP(3) receptors was markedly enhanced. These effects were reversed by expression of exogenous p97. In addition, Ufd1 and Npl4, which complex with p97, also associated with IP(3) receptors upon hormonal stimulation. We conclude that the p97-Ufd1-Npl4 complex couples ubiquitinated IP(3) receptors to proteasomal degradation and, thus, plays a key role in IP(3) receptor processing. These data also establish that the p97-Ufd1-Npl4 complex mediates endoplasmic reticulum-associated degradation in mammalian cells.
Subject(s)
Adenosine Triphosphatases/physiology , Calcium Channels/metabolism , Carrier Proteins/physiology , Endoplasmic Reticulum/metabolism , Nuclear Proteins/physiology , Proteins/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Adenosine Triphosphatases/metabolism , Animals , Calcium/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line , Cytosol/metabolism , DNA, Complementary/metabolism , Down-Regulation , Electrophoresis, Polyacrylamide Gel , Fibroblasts/metabolism , Immunoprecipitation , Inositol 1,4,5-Trisphosphate Receptors , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Mice , Nuclear Proteins/metabolism , Proteins/metabolism , RNA Interference , Rats , Subcellular Fractions , Time Factors , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
The extent to which chromosomal domains are reorganized within the nucleus during differentiation is central to our understanding of how cells become committed to specific developmental lineages. Spatio-temporal patterns of DNA replication are a reflection of this organization. Here, we demonstrate that the temporal order and relative duration of these replication patterns during S-phase are similar in murine pluripotent embryonic stem (ES) cells, primary adult myoblasts, and an immortalized fibroblast line. The observed patterns were independent of fixation and denaturation techniques. Importantly, the same patterns were detected when fluorescent nucleotides were introduced into living cells, demonstrating their physiological relevance. These data suggest that heritable gene silencing during commitment to specific cell lineages is not mediated by global changes in the sub-nuclear organization and replication timing of chromosome domains.
