To understand the function of γTuRC, it is important to identify all of its subunits and study their interactions. We report here the identification of Dgp71WD, a new γTuRC subunit that does not have grip motifs. We found that both Dgp71WD and γ-tubulin interacted directly with the subunits containing grip motifs. These findings shed new light on the structural organization of the γTuRC.
Sequence analyses suggested that there are two additional Drosophila
grip motif–containing proteins with predicted molecular masses of 79.8 kDa (AAF50536) and 223 kDa (AAF44968; Murphy et al., 2001
). The putative 79.8-kDa protein, which we will refer to as Dgrip79 hereafter, may correspond to one of the uncharacterized γTuRC subunits in the ~75-kDa molecular mass range (Figure A). However, the putative 223-kDa protein may not be an integral subunit of the Drosophila
γTuRC, because all the Drosophila
γTuRC subunits are smaller than 223 kDa. If Dgrip79 is a γTuRC subunit and if our estimation of a total of eight subunits in γTuRC is accurate, we may have identified a complete set of Drosophila
Interestingly, six (Dgrips75, 79, 84, 91, 128, and 163) of eight γTuRC subunits contain the conserved grip motifs. This sequence conservation suggests that the Dgrips interact with common proteins either within or outside of the γTuRC. We showed that, consistent with this idea, Dgrips84, 91, 128, and 163 directly interacted with γ-tubulin and Dgp71WD, two γTuRC subunits with no grip motifs (Gunawardane et al., 2000
and this study). It will be important to determine whether the grip motifs in the Dgrips mediate these interactions.
The finding that all Dgrips interact with γ-tubulin directly is intriguing because it suggests that the Dgrips75, 79, 128, and 163 that were originally thought to be the cap subunits, directly contact γ-tubulin in the γTuRC. If this is the case, the current γTuRC model, which hypothesizes that the γTuRC ring consists exclusively of γTuSCs (Oegema et al., 1999
; Moritz et al., 2000
), would need to be revised. We speculate that each of the Dgrips75, 79, 128, and 163 could form dimers with γ-tubulin molecules. These dimers along with several γTuSCs may be required to form the ring of the γTuRC.
Structural studies revealed that WD repeats form a β-propeller fold that could mediate protein-protein interactions (Smith et al., 1999
). We suggest that Dgp71WD could provide a scaffold via its WD-repeats to tether all of the Dgrips together in the γTuRC. Consistent with this idea, we found that four Dgrips (84, 91, 128, and 163) interact directly with Dgp71WD. Clearly, further structural and biochemical studies are necessary to determine whether some or all of the Dgrips75, 79, 128, and 163 participate in the formation of the γTuRC ring. Also, it is important to determine whether and how any of these Dgrips participate in the formation of the cap structure of the γTuRC.
We found that coexpressing any one of the grip motif–containing subunits, Dgrips84, 91, 128, and 163, with γ-tubulin was sufficient to promote γ-tubulin to bind to GTP. However, coexpressing γ-tubulin with Dgp71WD, which does not contain grip motifs, did not facilitate γ-tubulin binding to GTP. This finding showed that the interactions between γ-tubulin and Dgrips have a significantly different consequence from the interaction between γ-tubulin and Dgp71WD. Furthermore, it suggests that the grip motif–containing subunits play a role in regulating the GTP binding properties of γ-tubulin. Because GTP is important for α- and β-tubulin function, we suspect that Dgrips may be important for γ-tubulin function. Consistent with this idea, we found that γ-tubulin expressed alone could not incorporate into γTuRC in vitro; but coexpressing one of the Dgrips with γ-tubulin was sufficient for the incorporation (unpublished data).
Previously, human and Chlamydomonas
γ-tubulins expressed alone using rabbit reticulocyte lysate and baculovirus expression systems, respectively, were used to study the microtubule binding and nucleating activities of γ-tubulin (Li and Joshi, 1995
; Vassilev et al., 1995
; Leguy et al., 2000
). However, whether the γ-tubulins could bind to guanine nucleotides was not determined (Li and Joshi, 1995
; Vassilev et al., 1995
; Leguy et al., 2000
). Because we found that γ-tubulin expressed alone did not bind GTP, caution should be used in analyzing the function of γ-tubulin in the absence of Dgrips.
The interaction we observed between Dgp71WD and γ-tubulin should also be interpreted cautiously. Because γ-tubulin found in γTuRC and γTuSC can be cross-linked to GTP, the inability of γ-tubulin to bind to GTP when coexpressed with Dgp71WD may indicate that γ-tubulin does not assume the same structural conformation as in the γTuRC. One possibility is that the γ-tubulin coexpressed with Dgp71WD was not folded properly. If so, the interaction between Dgp71WD and γ-tubulin that we observed may not reflect a true interaction in the γTuRC. Alternatively, it is possible that the γ-tubulin expressed with Dgp71WD was folded correctly, but assumed a slightly different conformation to disfavor GTP binding in our in vitro assays. If so, the interaction between Dgp71WD and γ-tubulin is likely to reflect a true interaction in the γTuRC. Structural studies of γTuRC will be important to resolve whether γ-tubulin directly contact Dgp71WD in the complex.