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Cytoplasmic aggregates of ubiquitinated TAR DNA‐binding protein 43 (TDP‐43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP‐43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP‐43 on aggregate formation and fragmentation, using TDP‐43 expression constructs for WT or mutant TDP‐43 with a modified nuclear localizing signal (LQ‐NLS). Overexpression of the LQ‐NLS mutant alone induced no detectable cytoplasmic aggregates during a 72‐hr period. Polyubiquitination of both WT TDP‐43 and the LQ‐NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP‐43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ‐NLS mutant. Polyubiquitin‐charged WT …