The Mouse t-Complex-encoded Protein Tctex-1 Is a Light Chain of Brain Cytoplasmic Dynein

Abstract

19358 -19366). Peptide sequence data (16/16 residues correct) implicate the M r ‫؍‬ 14,000 polypeptide as Tctex-1, a protein encoded within the mouse t-complex. Tctex-1 cosediments with microtubules and is eluted with ATP or salt but not with GTP as expected for a dynein subunit. The ATP-eluted protein precisely cosediments with known cytoplasmic dynein proteins in sucrose density gradients. Tctex-1 also is immunoprecipitated from brain and other tissue homogenates by a monoclonal antibody raised against the 74-kDa cytoplasmic dynein intermediate chain. Quantitative densitometry indicates that Tctex-1 is a stoichiometric component of the dynein complex. As Tctex-1 is a candidate for involvement in the transmission ratio distortion (meiotic drive) of mouse t-haplotypes, these results suggest that cytoplasmic dynein dysfunction may play an important role in non-mendelian chromosome segregation.Dyneins are microtubule-based molecular motors. Axonemal dyneins are responsible for generation and propagation of ciliary/flagellar bends (see Ref. 1), whereas cytoplasmic dynein is involved in a wide range of intracellular motile events including retrograde vesicle transport in axons, membrane trafficking, nuclear migration, and both the positioning and anaphase movement of the mitotic spindle (2-9). Although involved in diverse motile systems, recent molecular analyses have revealed that the flagellar and cytoplasmic dynein isozymes are related both in terms of their component polypeptides and in their overall structural organization (for review, see Refs. 10 and 11).Cytoplasmic dynein is a multimeric complex containing two dynein heavy chains (DHCs) 1 (ϳ530 kDa) that exhibit both ATPase and motor activities; these components form the globular heads and stems seen in the soluble particle (reviewed in Ref. 10). A recent report suggests that there are at least three cytoplasmic DHC isoforms in mammals that are associated with discrete intracellular structures (12). Thus, different cytoplasmic DHC-containing isozymes may have distinct functions, as do their flagellar counterparts (reviewed in Refs. 11 and 13).Associated with these large motor subunits are 2-3 copies of a 74-kDa intermediate chain (IC74) (14) that contains five repeats of the WD motif (15) and is therefore likely a member of the ␤-propeller family of proteins (16). IC74 apparently is involved in attaching the cytoplasmic dynein motor to its cargo by mediating the interaction between dynein and dynactin (17, 18). Multiple alternatively spliced and phosphorylated variants of this component have been identified (14, 19 -21), and these may be involved in targeting the motor to various intracellular cargoes. The cytoplasmic ICs are related both structurally and functionally to their counterpart proteins found within flagellar dyneins (reviewed in Ref. 11). For example, the M r ϭ 78,000 IC (IC78) of Chlamydomonas outer arm dynein is also a WDrepeat protein that mediates the ATP-insensitive (cargo) attachment of that enzyme to the flagellar doublet microtubules (22...

A Novel Tctex2-related Light Chain Is Required for Stability of Inner Dynein Arm I1 and Motor Function in the Chlamydomonas Flagellum

“…a, MBP fusions to Tctex2b, Tctex1, and LC2 were digested with Factor Xa, and the LCs were separated from MBP by SDS-PAGE. Blots were probed with either affinity-purified rabbit polyclonal antibody CT117 (Tctex2b) or R5205 (Tctex1) (22). Both antibodies are specific and only recognize their respective LC.…”

“…Tctex2b Defines a New Subfamily of Dynein LCs-To date, three members of the Tctex1/Tctex2 family have been identified in Chlamydomonas; they are Tctex1, an inner arm I1 subunit (13) that also functions as a component of cytoplasmic dynein (22), LC2 (24), an outer arm LC that is required for assembly of that dynein (28), and Tctex2b, which is described here. Identification of Tctex2b allowed us to subdivide the Tctex2 family into two major groups.…”

“…This novel LC is the first Tctex2 protein to be identified within the inner arm system. Because Tctex1 is present in both inner arm I1 and cytoplasmic dynein (13,22) it will be interesting to determine whether Tctex2b also functions as a cytoplasmic dynein subunit.…”

“…The Tctex1 and Tctex2 genes were initially identified in mice within a 30 -40-megabase pair region of chromosome 17 referred to as the t-complex and are both candidates for distorter/sterility factors, which play roles in the non-Mendelian inheritance of variant forms of this chromosome known as the t-haplotypes (25,26). Tctex1 is also a component of cytoplasmic dynein (22) and participates in a variety of motor/cargo interactions that include an association with rhodopsin within the vertebrate photoreceptor (27). LC2 is a Tctex2 homologue (24) that is essential for the assembly of the Chlamydomonas outer dynein arm within the flagellar axoneme (28).…”

“…In Chlamydomonas, LC8 is also found in the outer arms and radial spokes and is required for intraflagellar transport (13,20,21). Tctex1 is a member of the Tctex1/Tctex2 family of dynein LCs (13,22) that includes rp3 (23) and the Chlamydomonas outer arm subunit LC2 (24). The Tctex1 and Tctex2 genes were initially identified in mice within a 30 -40-megabase pair region of chromosome 17 referred to as the t-complex and are both candidates for distorter/sterility factors, which play roles in the non-Mendelian inheritance of variant forms of this chromosome known as the t-haplotypes (25,26).…”