GENETIC INTERACTIONS AMONG CYTOPLASMIC DYNEIN, DYNACTIN, AND NUCLEAR-DISTRIBUTION MUTANTS OF NEUROSPORA-CRASSA

Cytoplasmic dynein is a multisubunit, microtubule-associated, mechanoc hemical enzyme that has been identified as a retrograde transporter of various membranous organelles. Dynactin, an additional multisubunit c omplex, is required for efficient dynein-mediated transport of vesicle s in vitro. Recently, we showed that three genes defined by a group of phenotypically identical mutants of the filamentous fungus Neurospora crassa encode proteins that are apparent subunits of either cytoplasm ic dynein or dynactin. These mutants, designated ropy (ro), display ab normal hyphal growth and are defective in nuclear distribution. We pro pose that mutations in other genes encoding dynein/dynactin subunits a re likely to result in a ropy phenotype and have devised a genetic scr een for the isolation of additional ro mutants. Cytoplasmic dynein/dyn actin is the largest and most complex of the cytoplasmic motor protein s, and the genetic system described here is unique in its potentiality for identifying mutations in undefined genes encoding dynein/dynactin subunits or regulators. We used this screen to isolate > 1000 ro muta nts, which were found to define 23 complementation groups. Unexpectedl y, interallelic complementation was observed with some allele pairs of ro-1 and ro-3, which are predicted to encode the largest subunits of cytoplasmic dynein and dynactin, respectively. The results suggest tha t the Ro1 and Ro3 polypeptides may consist of multiple, functionally i ndependent domains. In addition, approximate to 10% of all newly isola ted ro mutants display unlinked noncomplementation with two or more of the mutants that define the 23 complementation groups. The frequent a ppearance of ro mutants showing noncomplementation with multiple ro mu tants having unlinked mutations suggests that nuclear distribution in filamentous fungi is a process that is easily disrupted by affecting e ither dosage or activity of cytoplasmic dynein, dynactin, and perhaps other cytoskeletal proteins or regulators.