Valosin-containing protein (p97) is a regulator of endoplasmic reticulum stress and of the degradation of N-end rule and ubiquitin-fusion degradation pathway substrates in mammalian cells

Abstract

Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family (ATPases with multiple cellular activities) involved in multiple cellular functions, including degradation of proteins by the ubiquitin (Ub)-proteasome system (UPS). We examined the consequences of the reduction of VCP levels after RNA interference (RNAi) of VCP. A new stringent method of microarray analysis demonstrated that only four transcripts were nonspecifically affected by RNAi, whereas approximately 30 transcripts were affected in response to reduced VCP levels in a sequence-independent manner. These transcripts encoded proteins involved in endoplasmic reticulum (ER) stress, apoptosis, and amino acid starvation. RNAi of VCP promoted the unfolded protein response, without eliciting a cytosolic stress response. RNAi of VCP inhibited the degradation of R-GFP (green fluorescent protein) and Ub-(G76V)-GFP, two cytoplasmic reporter proteins degraded by the UPS, and of alpha chain of the T-cell receptor, an established substrate of the ER-associated degradation (ERAD) pathway. Surprisingly, RNAi of VCP had no detectable effect on the degradation of two other ERAD substrates, alpha1-antitrypsin and deltaCD3. These results indicate that VCP is required for maintenance of normal ER structure and function and mediates the degradation of some proteins via the UPS, but is dispensable for the UPS-dependent degradation of some ERAD substrates.

Figure 1.

RNAi of VCP induces ER stress and induction of UPR. HeLa cells were subjected to RNAi of VCP by using either VCP-2 or VCP-6 siRNA as described in Materials and Methods. (A) RT-PCR was performed for the indicated messages from cells subjected to RNAi and from cells treated for 6 h with 10 μM MG132, 10 μg/ml tunicamycin, and 5 μM brefeldin A. Arrows show spliced and unspliced transcripts of XBP-1. (B) Western blotting was performed for the indicated proteins from cells subjected to RNAi against VCP and from cells treated with 10 μM MG132 for 6 h. (C) Effect of RNAi on levels of VCP and polyubiquitinated proteins. HeLa cells were subjected to RNAi of VCP with the indicated siRNAs or treated with 10 μM MG132 for 6 h. Cell lysates were Western blotted for VCP (top) or polyubiquitin (bottom). Blots were quantified, and data are expressed as a percentage of control values. Results represent means ± SEM from five independent experiments.

Figure 2.

RNAi of VCP promotes vacuolization of cells. HeLa cells were transfected with siRNAs directed against EGFP (control) or VCP as described in Materials and Methods, or treated with MG132 for 6 h before processing for transmission electron microscopy. (A) Control cells. (B) MG132-treated cells. Arrows show aggregation of high-electron-density cytosolic material. (C) RNAi of VCP by using VCP-2 siRNA. (D) RNAi of VCP by using VCP-6 siRNA.

Figure 3.

Pharmacological induction of ER stress is not always associated with cell vacuolization. Immunofluorescence images of HeLa cells labeled by the FK1 anti-ubiquitin antibody (red) and the nuclear dye YoPro iodide (green). Cells were submitted to a 16 h treatment with 5 μM BFA and 10 μg/ml tunicamycin, either alone or in combination with 10 μM MG132. Although both MG132 and BFA induce cell vacuolization, probably by distension of ER cistaernae (arrows), tunicamycin induces an elongated cellular phenotype without inducing vacuoles, which can be induced by a cotreatment with MG132.

Figure 4.

Differential effects of RNAi of VCP on the degradation of UPS substrates. (A) Schematic representation of five UPS substrates used in this study. (B) Overexpression of UPS substrates does not induce UPR in stable cell lines as assessed by XBP-1 splicing. (C) HeLa cells stably