Dynactin subunit p150Glued isoforms notable for differential interaction with microtubules

Abstract

Dynactin is a multiprotein complex that enhances dynein activity. The largest dynactin subunit, p150Glued, interacts with microtubules through its N-terminal region that contains a globular cytoskeleton-associated protein (CAP)-Gly domain and basic microtubule-binding domain of unknown structure. The p150Glued gene has a complicated intron-exon structure, and many splice isoforms of p150Glued protein have been predicted. Here we describe novel natural 150 kDa isoforms: the p150Glued-1A isoform, whose basic domain is composed of 41 amino acids, and p150Glued-1B with a basic domain of 21 aa because of the lack of exons 5-7 in the corresponding messenger RNA (mRNA). According to reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot data, p150Glued-1A is expressed in nerve tissues, in cultured cells and in embryonic tissues, while 1B is expressed ubiquitously. Overexpression of GFP-p150Glued-1A and -1B fusion proteins and immunostaining of cultured cells with 1A-specific antibodies show that the p150Glued-1A isoform is distributed along microtubules, whereas 1B is associated with microtubule plus-ends. The higher affinity of the p150Glued-1A isoform for microtubules is confirmed by a co-pelleting assay. In fibroblast-like cells, the interaction of p150Glued-1A with microtubules is less dependent on EB1/EB3 and CLIP170 proteins, compared with p150Glued-1B. In polarized cells, p150Glued-1A decorates microtubules that face the leading edge of the cell. The pattern of p150Glued-1A and p150Glued-1B interaction with microtubules and their tissue-specific expression patterns suggest that these isoforms might be involved in cell differentiation and proliferation. © 2009 John Wiley & Sons A/S.