Biochemical and Molecular Analysis of the Mammalian Cytoplasmic Dynein Intermediate Chain

Abstract

Cytoplasmic dynein is a multisubunit protein complex responsible for the intracellular movement of membranous organelles and other cargo along microtubules. The heavy chains contain the motor domains, while the intermediate chain and other subunits are important for binding to cargo. There are at least five different intermediate chain polypeptides, the products of alternative splicing of two genes. The cytoplasmic dynein intermediate chains are also phosphorylated. The expression of the different intermediate chain mRNAs is characterized by reverse transcription-polymerase chain reactions using oligonucleotide primers appropriate for the alternative splicing sites. The presence of the different intermediate chain polypeptide isoforms is determined by two-dimensional gel analysis of cytoplasmic dynein samples. The phosphorylation state of the polypeptides is determined by treatment of immunoprecipitated cytoplasmic dynein with protein phosphatase and analysis of changes in polypeptide spot distribution after two-dimensional gel electrophoresis.