A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

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O R I GI N A L P A P E R

P. F. Minke á I. H. Lee á J. H. Tinsley á M. Plamann

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

Received: 2 October 1999 / Accepted: 7 June 2000 / Published online: 21 July 2000 Ó Springer-Verlag 2000

Abstract Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identi®ed as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural sca€old protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the ®lamentous fungus Neurospora crassa, we have identi®ed genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding. Communicated by C. A. M. J. J. van den Hondel I. H. Lee á M. Plamann (&) School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110-2499, USA E-mail: [email protected] Tel.: +1-816-2352593 Fax: +1-816-2351503 P. F. Minke á J. H. Tinsley1 Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA Present address: 1 Texas A&M University Health Science Center, 1901 South First Street, Building 4, Temple, TX 76504, USA

Key words Cytoplasmic dynein á Dynactin á p150Glued á Arp1 á Microtubules

Introduction The actin-related proteins (Arps) comprise a large family of proteins whose members show varying degrees of sequence identity to the cytoskeletal protein actin, yet are suciently divergent to prevent their being classi®ed as actin isoforms (for reviews see Frankel and Mooseker 1996; Schafer and Schroer 1999). Members of the Arp1 class are most similar to actin in amino acid sequence ± having 55% identity to conventional actin isoforms ± while members of the classes Arp2±11 are progressively less similar to actin. Arp1 is the major structural protein of dynactin, a multisubunit complex required for organelle transport by the microtubule-associated motor cytoplasmic dynein (for reviews see Allan 1996; Schroer 1996; Schroer et al. 1996; Holleran et al. 1998). Ultrastructural analysis of puri®ed dynactin reveals that Arp1 exists as a 37-nm actin-like ®lament consisting of eight to