Figure 6

ALS-associated CREST mutation Q388stop increases steady-state levels of the protein and its ability to aggregate. (A) A map of CREST with the positions of single amino acid substitutions or a deletion found in ALS indicated. (B) All CREST variants are localized predominantly to the nucleus in SH-SY5Y cells. (C) Q388stop mutant aggregates in the nucleus more often compared to the normal protein and other mutants. Among cells expressing each of the untagged CREST variants those with aggregates in the nucleus were quantified on the entire coverslip 24 hours post-transfection and obtained values were normalized against the value for wild-type CREST. The bar chart shows means ± s.e.m. of these normalized values from four independent experiments (*p < 0.05). (D) Steady-state protein levels for Q388stop mutant variant are increased compared to the wild-type protein without significant difference at the transcript level. The bar charts show means ± s.e.m. of seven independent experiments (*p < 0.05, Mann-Whitney U-test). (E) All CREST variants are stable proteins with half-lives of approximately 36 hours. Cycloheximide was used to block protein synthesis; levels of a short-lived protein, cyclin A, were measured to confirm successful block of translation. (F) Solubility of mutant CREST variants upon sequential protein extraction was comparable to that of wild-type protein. Fractionation was performed as described in Materials and methods. (G) CREST mutants do not affect the ability of the protein to be recruited to stress granules. The fraction of cells with CREST-positive stress granules after sodium arsenite exposure was quantified in the population of CREST-GFP expressing cells for each variant (at least 100 cells counted per variant in each of the two independent experiments). Scale bar, 10 μm.