ABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0046879.].
ABSTRACT
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 wild-type (p97) or mutant p97 (p97(R155C)) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9(KO)) cells. Native gel electrophoresis showed that both p97 and p97(R155C) assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97(R155C)-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 ATG9(KO)/p97(R155C)-RFP and ATG9(KO) cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9(KO) mutant. A major finding is that the expression of p97(R155C)-RFP in the ATG9(KO) 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.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Dictyostelium/metabolism , Point Mutation , Protein Multimerization , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Adenosine Triphosphatases/genetics , Autophagy/radiation effects , Cell Cycle Proteins/genetics , Chemotaxis/radiation effects , Dictyostelium/cytology , Dictyostelium/enzymology , Dictyostelium/genetics , Gene Knockout Techniques , Humans , Light , Proteasome Endopeptidase Complex/metabolism , Protein Multimerization/radiation effects , Protein Structure, Quaternary , Ubiquitination/radiation effects , Valosin Containing ProteinABSTRACT
Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell-autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP-tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9-GFP-containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9-deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild-type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.
