Heteromeric p97/p97R155C Complexes Induce Dominant Negative Changes in Wild-Type and Autophagy 9-Deficient Dictyostelium strains

et al. (2012) Heteromeric p97/p97R155C Complexes Induce Dominant Negative Changes in Wild-Type and Autophagy 9-Deficient Dictyostelium strains. PLoS ONE 7(10): e46879. doi:10.1371/journal.pone.0046879 R155C Heteromeric p97/p97 Complexes Induce Dominant Negative Changes in Wild-Type and Autophagy 9- Deficient Dictyostelium strains Khalid Arhzaouy 0 1 Karl-Heinz Strucksberg 0 1 Sze Man Tung 0 1 Karthikeyan Tangavelou 0 1 Maria Stumpf 0 1 Jan Faix 0 1 Rolf Schro der 0 1 Christoph S. Clemen 0 1 Ludwig Eichinger 0 1 Neeraj Vij, Johns Hopkins School of Medicine, United States of America 0 Current address: Hertie-Institute for Clinical Brain Research, University of Tu bingen , Tu bingen , Germany 1 1 Intitute for Biochemistry I, Medical Faculty, University of Cologne , Cologne, Germany , 2 Institute of Neuropathology, University Hospital Erlangen , Erlangen, Germany , 3 Institute for Biophysical Chemistry, Hannover Medical School , Hannover , Germany Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wildtype (p97) or mutant p97 (p97R155C) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9KO) cells. Native gel electrophoresis showed that both p97 and p97R155C assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97R155C-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9KO/ p97R155C-RFP and ATG9KO cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9KO mutant. A major finding is that the expression of p97R155C-RFP in the ATG9KO strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition. - Funding: The work was supported by grants from the German Research Foundation(DFG-FOR1228: SCHR 562/9-1, CL 381/3-1, EI 399/5-1) awarded to RS, CC and LE as well as from Ko ln Fortune awarded to CC and LE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. " These authors also contributed equally to this work. The late-onset autosomal dominant multisystem disorder IBMPFD is caused by mutations of the human p97 (synonyms: valosin containing protein (VCP) or TER ATPase in mammals, TER94 in Caenorhabditis elegans, Cdc48p in yeast, VAT in archaebacteria) gene on chromosome 9p13-p12 [1,2]. At least 20 unique p97 missense mutations cause either IBMPFD [35], ALS14 [6], or HSP [7,8] with codon 155 being a mutation hot spot. p97 is a ubiquitously expressed and evolutionarily highly conserved member of the AAA-ATPase family (ATPases Associated with a wide variety of cellular Activities). The protein has a tripartite structure comprising an N-terminal domain (CDC48) involved in ubiquitin binding, and two central D1 and D2 domains which bind and hydrolyze ATP [9]. p97 assembles into functional hexamers with the D domains forming a central cylinder, which is surrounded by the N-terminal domains [10]. In protein quality control and protein homeostasis p97 is a key player in endoplasmic reticulum associated protein degradation (ERAD), the ubiquitin proteasome protein degradation system (UPS), aggresome formation and autophagosome maturation [1117]. Macroautophagy (hereafter autophagy) is an ancient cellular pathway to recycle cellular material that is conserved from yeast to man [18,19]. More than 30 autophagy (ATG) genes have been identified, mainly in yeast, of which 18 constitute the core machinery for starvation induced autophagy. Autophagy contributes to many physiological and pathological processes, including cell differentiation and development, programmed cell death, cancer and neurodegenerative disorders [20]. The use of model organisms, such as Saccharomyces cerevisiae, C. elegans, Drosophila melanogaster or D. discoideum, in the study of the cellular consequences of mutations that cause human disease offers a number of advantages and has steadily increased in recent years. Disease-causing mutations can only be studied in a very limited way in patients, and even in mouse models their analysis is usually expensive, time consuming and technically challenging or sometimes even impossible. In contrast, their functional analysis in D. discoideum and other simple model organisms is often easier, faster and cheaper [21,22]. Despite its lower complexity, D. discoideum is very similar to higher eukaryotes in many cellular aspects and for example is increasingly used to study autophagy and human disease genes [23,24]. A major advantage of Dictyostelium is a large toolbox for the generation of mutants [25]. Previous work in D. discoideum showed that autophagy is required for normal development. Autophagy mutants were generated in six core autophagy genes and all mutants displayed developmental defects albeit of variable severity [26,27]. ATG9 deficient cells had a pleiotropic phenotype and displayed severe defects in growth, phagocytosis and development [28]. Here we report on the analysis of D. discoideum strains that ectopically express p97 as well as p97R155C fused to RFP in AX2 wild-type and ATG9KO cells. The AX2/p97R155C-RFP strain mirrors the situation in heterozygous patients, while ATG9KO/ p97R155C-RFP cells allow the investigation of mutant p97 in an autophagy deficient background. We provide genetic, biochemical, and cell biological evidence that p97 functionally links proteasomal activity and autophagy in Dictyostelium. Materials and Methods Dictyostelium Strains, Growth, Development, and Phototaxis D. discoideum strain AX2 was used as wild-type strain. Generation of ATG9 knock-out cells has been described previously [28]. Strains expressing p97-RFP and p97R155C-RFP were generated by transformation of AX2 and ATG9KO cells [28], respectively, with an expression construct encoding the fusion protein in the p389-2 mRFPmars vector [29]. Wild-type and mutant strains were grown at 21uC in liquid nutrient medium on plates (90 mm diameter) or with shaking at 1 (...truncated)