Dynactin subunit p150Glued isoforms notable for differential interaction with microtubules.
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.
Zhapparova
Olga N
ON
Institute of Protein Research of Russian Academy of Sciences, 34 Vavilova Str., Moscow 117334, Russia.
Bryantseva
Sofiya A
SA
Dergunova
Liudmila V
LV
Raevskaya
Natalia M
NM
Burakov
Anton V
AV
Bantysh
Olga B
OB
Shanina
Nina A
NA
Nadezhdina
Elena S
ES
D000595:Amino Acid Sequence; D016764:Cell Polarity / Q000235:genetics; D002478:Cells, Cultured; D000072159:Dynactin Complex; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins