Functional proteome analysis of pathophysiological variants of the AAA-ATPase Cdc48p, VCP

The yeast ‘cell division cycle protein 48’ (Cdc48p) and its highly conserved human orthologue ‘valosin-containing protein’ (VCP) are ubiquitin-controlled AAA-ATPases (ATPases associated with various cellular activities). Cdc48p/VCP is involved in different cellular processes, such as ER-associated protein degradation (ERAD), membrane fusion and cell cycle control. VCP has been proposed as a sensor for misfolded proteins associated with neurodegenerative disorders, such as Machado-Joseph, Parkinson and Alzheimer disease. Upon mutation human VCP is causative for ‘inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia’ (IBMPFD), a hereditary disease characterized by protein deposits in either myofibrils or brain. Mutation of Cdc48p/VCP leads to apoptotic cell death in mammalian cells, in zebrafish, Caenorhabditis elegans and in the unicellular organism Saccharomyces cerevisiae. However, the cellular mechanisms of apoptotic cell death upon mutation of Cdc48p/VCP and the pathophysiological role of this protein in the progression of human degenerative disorders have remained unresolved. In this study, a systematic proteome analysis of the apoptotic cdc48S565G mutant yeast strain was performed, in order to reveal apoptosis-associated proteome alterations that may help to elucidate cellular mechanisms underlying Cdc48p/VCP-mediated cell death. Differential two-dimensional gel electrophoresis (2-DE) of the apoptotic cdc48S565G mutant and the CDC48 wild-type yeast strain resulted in significant protein spot alterations in mitochondrial extracts. Both altered amounts of mitochondrial proteins and accumulation of Cdc48p-S565G itself and other proteins associated with the NE-ER (nuclear envelope-endoplasmic reticulum) were observed in mitochondrial extracts of the cdc48S565G strain. Starting from these results, mitochondria were demonstrated to play a pivotal role during apoptotic cell death in the cdc48S565G strain: Depletion of mitochondrial proteins essential for mitochondrial function was found to be associated with mitochondrial swelling, respiratory deficiency of yeast cells, release of cytochrome c into the cytosol, and accumulation of reactive oxygen species (ROS) produced by mitochondria. Generating yeast strains completely lacking functional mitochondria (ρ0 strains) resulted in the assimilation of the viabilities of wild-type and cdc48S565G cells under apoptotic growth conditions, confirming a deleterious role of mitochondria during apoptotic cell death. The increased co-purification of Cdc48p-S565G and other NE-ER-associated proteins with mitochondria as observed by 2-DE was demonstrated to be due to an augmented co-migration of microsomes with mitochondria in mitochondrial extracts. Ultrastructural analysis by electron microscopy revealed augmented proximity of NE-ER membranes and mitochondria in cdc48S565G cells, indicating an increased association of NE-ER and mitochondria in apoptotic cdc48S565G cells. Increased amounts of polyubiquitinated proteins in total cell extracts and microsomal extracts point to a dysfunction of Cdc48p-S565G in ERAD. Polyubiquitinated proteins were found to be strongly associated with a subset of mitochondria, whereas the majority of mitochondria were not affected. The polyubiquitinated mitochondrial fraction comprised markedly increased amounts of Cdc48p-S5656, ER stress marker Kar2p and microsomes. The observed increased association of NE-ER and mitochondria in cdc48S565G cells demonstrates for the first time that cellular stress localized at the NE-ER due to mutation in CDC48 and dysfunction of ERAD is transmitted to mitochondria. This may result in mitochondrial damage that initiates mitochondria to produce ROS and thus triggering apoptotic cell death. In order to verify if the results obtained in the cdc48S565G yeast strain are relevant for events in VCP-mediated cell death, a mammalian cell culture system expressing human VCP variants was established. Decreased cell viability, increased apoptotic cell death and accumulation of polyubiquitinated proteins was observed in cells expressing the dominant ATPase-deficient human VCP variant VCP-K524A and the VCP variant VCP-S555G, the homologous variant to yeast Cdc48p-S565G. Coexpression of VCP and of the ERAD substrate rhodopsin resulted in the accumulation of aggregated rhodopsin in the case of VCP-K524A and VCP-S555G, showing the defect of these variants in ERAD. Since these are highly similar features as observed in the cdc48S565G yeast strain, these data obtained in mammalian cell culture validate the use of the yeast model system. In contrast, in mammalian cells expression of the IBMPFD-causing VCP variant VCP-R155H did not affect cell viability. However, VCP-R155H demonstrated deficiency in ERAD but much less severe comparerd to VCP-K524A and VCP-S555G. Thus, ERAD deficiency upon Cdc48p/VCP mutation is per se not sufficient to trigger cell death. However, the ERAD deficiency of VCP-R155H might become deleterious and may result in the induction of apoptotic cell death upon enrichment of proteins during ageing. Based on the results obtained in the cdc48S565G yeast strain, a crucial mitochondrial involvement during apoptosis in cells with dysfunctional VCP and ERAD should be evaluated in the future.