Association of cytoplasmic dynein 1b/2 and the major flagellar membrane glycoprotein, FMG-1B

Ewelina Betleja1, Robert A. Bloodgood2, Malgorzata Zurawska1, and Douglas G. Cole1

1) Dept. Microbiol, Molec Biol & Biochem, Univ of Idaho, Moscow, ID 83844, USA 2) Dept. Cell Biol, Univ of Virginia School of Medicine, Charlottesville, VA 22908, USA

Consisting of the bi-directional movement of large protein particles, intraflagellar transport (IFT) is required for the assembly and function of eukaryotic cilia and flagella. The anterograde movement of particles is driven by kinesin-2 motors while retrograde IFT is powered by cytoplasmic dynein 1b/2, also known as IFT dynein. How the IFT particles dock onto these motors is not well understood and may involve, as yet unidentified, adaptor proteins that bridge motor and transport complex. In an attempt to identify specific IFT motor interactors, we designed a pull-down assay using recombinant dynein heavy chain (MBP-DHC1b). Although it failed to bind IFT complex A or B, the DHC1b resin specifically down other proteins from Chlamydomonas flagellar membrane plus matrix (M+M). Mass spectrometry identified two of these as the major flagellar membrane glycoprotein, FMG-1B, and a second protein, temporarily named cytoplasmic dynein 1b/2-associated protein (CDAP). To verify the association of these proteins, FMG-1B was immunoprecipitated from M+M using anti-FMG-1B resin. Co-precipitating with the FMG-1B was CDAP and two of the dynein subunits, DHC1b and D1bLIC. Consistent with this observation, sucrose density gradient centrifugation of M+M revealed partial co-sedimentation of dynein 1b/2, FMG-1B and CDAP. To address in vivo associations, we screened retrograde IFT mutants with known disruptions in dynein genes (dhc1b, d1blic & fla14) and identified marked reduction of whole cell CDAP in each strain. Thus, cytoplasmic dynein 1b/2 appears to be required for the stability of CDAP. Since FMG-1B functions in gliding, we are currently testing the hypothesis that cytoplasmic dynein 1b/2 works with CDAP and FMG-1B to generate gliding forces. Supported by GM61920 (DGC) and P20RR016454 (DGC).

e-mail address of presenting author: ebetleja@vandals.uidaho.edu