Abstract
Familial amyotrophic lateral sclerosis type 2 (ALS2) is a juvenile autosomal recessive motor neuron disease caused by the mutations in the ALS2 gene. The ALS2 gene product, ALS2/alsin, forms a homophilic oligomer and acts as a guanine nucleotide-exchange factor (GEF) for the small GTPase Rab5. This oligomerization is crucial for both Rab5 activation and ALS2-mediated endosome fusion and maturation in cells. Recently, we have shown that pathogenic missense ALS2 mutants retaining the Rab5 GEF activity fail to properly localize to endosomes via Rac1-stimulated macropinocytosis. However, the molecular mechanisms underlying dysregulated distribution of ALS2 variants remain poorly understood. Therefore, we sought to clarify the relationship between intracellular localization and oligomeric states of pathogenic ALS2 variants. Upon Rac family small GTPase 1 (Rac1) activation, all mutants tested moved from the cytosol to membrane ruffles but not to macropinosomes and/or endosomes. Furthermore, most WT ALS2 complexes were tetramers. Importantly, the sizes of an ALS2 complex carrying missense mutations in the N terminus of the regulator of chromosome condensation 1-like domain (RLD) or in-frame deletion in the pleckstrin homology domain were shifted toward higher molecular weight, whereas the C-terminal vacuolar protein sorting 9 (VPS9) domain missense mutant existed as a smaller dimeric or trimeric smaller form. Furthermore, in silico mutagenesis analyses using the RLD protein structure in conjunction with a cycloheximide chase assay in vitro disclosed that these missense mutations led to a decrease in protein stability. Collectively, disorganized higher structures of ALS2 variants might explain their impaired endosomal localization and the stability, leading to loss of the ALS2 function.
Highlights
Familial amyotrophic lateral sclerosis type 2 (ALS2) is a juvenile autosomal recessive motor neuron disease caused by the mutations in the ALS2 gene
Upon Rac family small GTPase 1 (Rac1) activation, ALS2WT was relocalized to the surface of large vesicular compartments, whereas the majority of variant proteins remained in the cytoplasm (Fig. 1A)
Despite that pathogenic ALS2 variants failed to localize to vesicular compartments, a significant portion of them was recruited to the Rac1-induced F-actin– positive membrane ruffles (Fig. 1A), reproducing the phenotypes of two previously examined ALS2 variants: ALS2C157Y and ALS2G540E (Fig. S2B)
Summary
We have demonstrated that the majority of the ectopically expressed WT ALS2 protein (ALS2WT) is scattered throughout the cytoplasm in HeLa and COS-7 cells [10]. ALS2R1611W was broadly eluted in fractions with apparent molecular masses ranging from ϳ400 to ϳ700 kDa and an additional single minor peak of ϳ1000 kDa (Fig. 4, C and D), which was similar to those of ALS2L1617A mutant These results demonstrate that nonpathogenic ALS2 variants (ALS2P132L and ALS2E1173K) as well as the Rab GEF-defective artificial mutant (ALS2P1603A) primarily exist as tetrameric forms like ALS2WT. Quantitative analysis revealed that the total signal intensities in high-molecular weight (HMW) fractions (fractions 1– 8) for these mutants were comparable with that for ALS2–RLDWT, tetra-octameric fractions (fractions 9 –16) for ALS2–RLDS100I, ALS2–RLDP132L, and ALS2–RLDC157Y were significantly higher than that for ALS2–RLDWT with a concomitant decrease in their dimeric fractions (fractions 17–23) (Fig. 6C) These data indicate that some pathogenic missense mutations as well as normal variation in the N-terminal RLD can alter the oligomeric states of the ALS2–RLD fragment. These results are fully consistent with the previously reported model, which was built based upon the human RCC1 sequence as a template [62]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.