Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains.
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.
Intitute for Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany.
Khalid
K
Arhzaouy
Karl-Heinz
KH
Strucksberg
Sze Man
SM
Tung
Karthikeyan
K
Tangavelou
Maria
M
Stumpf
Jan
J
Faix
Rolf
R
Schröder
Christoph S
CS
Clemen
Ludwig
L
Eichinger
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis / Q000528:radiation effects; D004023:Dictyostelium / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006801:Humans; D008027:Light; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055503:Protein Multimerization / Q000528:radiation effects; D020836:Protein Structure, Quaternary; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D054875:Ubiquitination / Q000528:radiation effects; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D015800:Protozoan Proteins; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein