Unstable mutants in the peripheral endosomal membrane component ALS2 cause early-onset motor neuron disease - PubMed (original) (raw)

Unstable mutants in the peripheral endosomal membrane component ALS2 cause early-onset motor neuron disease

Koji Yamanaka et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Mutations in ALS2, carrying three putative guanine exchange factor (GEF) domains, are causative for a juvenile, autosomal recessive form of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and infantile-ascending hereditary spastic paralysis. Endogenous ALS2 is shown here to be enriched in nervous tissue and to be peripherally bound to the cytoplasmic face of endosomal membranes, an association that requires the amino-terminal "RCC1 (regulator of chromatin condensation)-like" GEF domain. Disease-causing mutants and a naturally truncated isoform of ALS2 are shown to be rapidly degraded when expressed in cultured human cells, including lymphocytes derived from patients with ALS2 mutations. Thus, mutations in the ALS2 gene linked to early-onset motor neuron disease uniformly produce loss of activity through decreased protein stability of this endosomal GEF.

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Figures

Fig. 1.

Endogenous WT ALS2 is enriched in neuronal tissues and membrane-containing fractions. (A) Predicted domain structures of ALS2 long (WT) and short form with reported sites for disease-associated mutations. The two antigens (ALS2RCC1, ALS21082) used for antibody production are also indicated. DH, Dbl homology; PH, pleckstrin homology; MORN, membrane occupation and recognition nexus. (B and C) Immunoblots of mouse tissue extracts and COS cell extracts. Aliquots of 70 μg of various mouse tissue extracts together with 5 μgof untransfected (COS-mock) and WT ALS2-transfected COS cell (COS-WT) lysates were immunoblotted as indicated. (D) Immunoblots of mouse nervous tissue extracts. Aliquots of 100 μg of mouse neuronal tissue extract and 5 μg of ALS2-transfected COS cell lysate were immunoblotted as indicated. (E) Relative abundance of endogenous ALS2 in mouse brain. Indicated amounts of mouse brain lysate and recombinant ALS2 (His-ALS2) were immunoblotted with pAb-ALS2RCC1. (F and G) Subcellular fractionation of mouse and human neuronal tissues as illustrated. Equal proportions of fractionated mouse brain and spinal cord (F) or human cerebral cortex and 0.2 times of the cytosol fraction (G) were immunoblotted as indicated. Tfn receptor, transferrin receptor; ER, endoplasmic reticulum.

Fig. 5.

ALS2 protein is not accumulated in lymphoblasts of ALS2 patients. Immunoblot analysis of lymphoblasts from ALS2 patients and a nondiseased patient control. Aliquots of 40μg of lymphoblast lysates and 0.5μg of transfected COS cell lysates were resolved with SDS/PAGE and blotted with pAb-ALS21082 (A) and pAb-ALS2RCC1 (B), respectively. WT ALS2 is indicated. The asterisk (*) denotes a nonspecific ≈120-kDa band. Both blots were reprobed with anti-α-tubulin antibody to demonstrate equal loading.

Fig. 2.

Endogenous WT ALS2 is a peripherally associated endosomal membrane protein oriented toward the cytoplasm. (A_–_C) Total membranes were processed for flotation centrifugation, alkali extraction, and proteinase sensitivity assays as illustrated. (A) Endogenous ALS2 is detected primarily in endosomal membrane fractions. After centrifugation, the membrane-containing interfaces and an aliquot of the layer containing soluble proteins (40%) were collected and immunoblotted as indicated. (B) Endogenous ALS2 is peripherally associated with intracellular membranes. After alkali extraction, aliquots of supernatants (S) and membrane-containing pellets (P) were immunoblotted as indicated. (C) Endogenous ALS2 resides on the cytoplasmic face of intracellular membranes. Aliquots of total membranes were treated with proteinase K in the presence or absence of detergent and immunoblotted as indicated. (D_–_G) Transfected ALS2 colocalizes with the endosomal markers EEA1 and transferrin receptor (TfR). Representative deconvolved images of COS cells transiently transfected with WT ALS2 (FITC, green, Top) and stained for endogenous membrane markers (Cy5, red, Middle): EEA1 (D), TfR (E), lysosome-associated membrane protein-2 (LAMP-2) (F), and protein disulfide isomerase (PDI) (G). Merged images together with enlarged boxed area that demonstrates merged (Left), ALS2 (green, Center), and endogenous marker (red, Right) images are shown (Bottom). (Magnifications: ×600.)

Fig. 3.

An intact amino-terminal RCC1-like domain is required for correct subcellular localization. (A) Schematic representation of the disease-associated ALS2 mutant and amino-terminal deletion expression constructs. Expression was verified by immunoblotting transiently transfected COS cell lysates as indicated. DH, Dbl homology; PH, pleckstrin homology; MORN, membrane occupation and recognition nexus. (B) Representative deconvolved micrographs of COS cells transiently transfected with indicated constructs and stained with pAb-ALS2RCC1 and FITC-conjugated goat anti-rabbit IgG. (C_–_I) Subcellular localization of ALS2 depends on an intact amino-terminal RCC1 domain. (C) HEK293 cells transiently transfected with amino-terminal-deleted ALS2 mutants were fractionated (see scheme in Fig. 1_F_) and immunoblotted with anti-FLAG M2 antibody. (D_–_I) Representative deconvolved images of COS cells transiently transfected with indicated constructs and stained with FITC-conjugated anti-FLAG M2 antibody (green, D_–_I) and endogenous (red) EEA1 (H) or transferrin receptor (TfR) (I). (Magnifications: ×600.)

Fig. 4.

Disease-causing ALS2 mutants and the short form of ALS2 are unstable proteins. (A) ALS2 mutant proteins are unstable compared with the WT ALS2 protein. Mutant ALS2 was transiently transfected in HEK293 cells in the presence or absence of 20 μM MG132 for 10 h. Lysates were analyzed by immunoblot with anti-FLAG M2 antibody (Upper). The same membrane was blotted with anti-α-tubulin antibody to demonstrate equal loading (Lower). (B and C) Half-lives of ALS2 and mutant proteins. Transfected HEK293 cells were metabolically radiolabeled, immunoprecipitated, and analyzed by SDS/PAGE (B). The radiolabeled ALS2 bands were quantified by densitometry and plotted. Calculated half-lives of the ALS2 proteins are as indicated (C).

References

1. Ben Hamida, M., Hentati, F. & Ben Hamida, C. (1990) Brain 113, 347-363. - PubMed
1. Hadano, S., Hand, C. K., Osuga, H., Yanagisawa, Y., Otomo, A., Devon, R. S., Miyamoto, N., Showguchi-Miyata, J., Okada, Y., Singaraja, R., et al. (2001) Nat. Genet. 29, 166-173. - PubMed
1. Yang, Y., Hentati, A., Deng, H. X., Dabbagh, O., Sasaki, T., Hirano, M., Hung, W. Y., Ouahchi, K., Yan, J., Azim, A. C., et al. (2001) Nat. Genet. 29, 160-165. - PubMed
1. Eymard-Pierre, E., Lesca, G., Dollet, S., Santorelli, F. M., Di Capua, M., Bertini, E. & Boespflug-Tanguy, O. (2002) Am. J. Hum. Genet. 71, 518-527. - PMC - PubMed
1. Gros-Louis, F., Meijer, I. A., Hand, C. K., Dube, M. P., MacGregor, D. L., Seni, M. H., Devon, R. S., Hayden, M. R., Andermann, F., Andermann, E., et al. (2003) Ann. Neurol. 53, 144-145. - PubMed

Unstable mutants in the peripheral endosomal membrane component ALS2 cause early-onset motor neuron disease - PubMed (original) (raw)