The γ-tubulin complex is required for microtubule nucleation at the centrosome. To determine the composition of the human γ-tubulin complex, we purified the complex from cultured cells and performed mass spectrometry analysis of peptides derived from the constituent proteins. We found the four previously characterized members of the human γ-tubulin complex, γ-tubulin, GCP2, GCP3, and GCP4, and identified two new components, GCP5 and GCP6. Although the cytoplasmic form of the γ-tubulin complex was purified and characterized, all of the known components also localize with γ-tubulin to the centrosome in vivo and bind with γ-tubulin to microtubules in vitro. With the identification of GCP5 and GCP6, all of the major proteins in the human γ-tubulin complex have now been identified. Here we consider the implications of these results for γ-tubulin function.
Current ideas for the mechanism of γ-tubulin complex function are based in part on its morphology. Frog (Zheng et al., 1995
), fly (Oegema et al., 1999
), and human (this study) γ-tubulin complexes all share a characteristic ring, or spiral, shape and are ~25 nm in diameter. This conservation of morphology strongly suggests that size and shape of the complex are important for function. Two models have been proposed for how the γ-tubulin complex nucleates microtubule polymerization. In the protofilament model, nucleation would occur by lateral interaction of α-tubulin/β-tubulin heterodimer subunits with a linear protofilament of γ-tubulin (Erickson and Stoffler, 1996
). When not actively engaged with a microtubule, the linear γ-tubulin protofilament is proposed to adopt a ring conformation, similar to GDP-tubulin protofilaments from depolymerizing microtubules. In the template model, nucleation would occur by head-to-tail interaction of γ-tubulin with α-tubulin/β-tubulin heterodimers. Each of the 13 protofilaments of a typical microtubule would thus be templated directly by one of the repeated ring subunits of the γ-tubulin complex, (Zheng et al., 1995
). Structural studies of the Drosophila
(Moritz et al., 2000
) and Xenopus
(Keating and Borisy, 2000
; Zhang et al., 2000
) γ-tubulin complexes and their interaction with microtubules best fit the template model. Our results on the size and shape of the human γ-tubulin complex are also most consistent with the template model. It seems unlikely that the diameter and subunit structure of the γ-tubulin complex would be conserved to approximate those of the microtubule were it not for functional constraints.
γ-Tubulin, GCP2, and GCP3 are conserved in all eukaryotes and thus are likely to form a core unit of microtubule nucleation. However, neither γ-tubulin alone (Leguy et al., 2000
) nor a γ-tubulin/GCP2/GCP3 small complex (Oegema et al., 1999
) are able to nucleate microtubules efficiently, indicating that the other γ-tubulin complex components are important for nucleation. It is interesting that S. cerevisiae
lacks GCP4, GCP5, and GCP6; it is likely that interactions with other proteins at the yeast spindle pole body serve the same function as these GCP components (reviewed by Jeng and Stearns, 1999
). Although it is possible to clearly identify orthologues of γ-tubulin, GCP2, and GCP3 in all eukaryotes, the other GCPs are less well conserved. For example, the orthologue relationships of vertebrate GCP4, GCP5, and GCP6 to the GCP-like proteins in plants are not clear.
Why are so many related proteins found in the γ-tubulin complex? The GCP components share sequence similarity in restricted domains, and it seems likely that these conserved domains share common functions. This is the case for other complexes composed of related proteins, such as the CCT chaperonin complex (also known as the TCP-1 complex or TriC) and the RF-C DNA replication factor (also known as activator 1). CCT is composed of eight different subunits that shares conserved domains. The conserved domains of the CCT proteins are involved in the ATPase activity of each of the subunits and may also be involved in interactions between subunits (Kim et al., 1994
). RF-C is composed of five subunits that shares eight regions of sequence similarity (reviewed by Mossi and Hubscher, 1998
). These regions are involved in complex formation and substrate binding. By analogy, the regions of sequence similarity in the GCPs might have a role in folding of the proteins to the correct conformation or in GCP-GCP subunit interactions within the complex.
Although the GCPs are related, we suspect that they are not redundant in function. This is based on the finding that GCP2/Spc97p and GCP3/Spc98p are both essential proteins in yeast (Geissler et al., 1996
; Knop et al., 1997
) and that the proteins in the vertebrate γ-tubulin complexes are present in unique stoichiometries (Murphy et al., 1998
; Zheng et al., 1995
; this study). Again, this is similar to the CCT complex, in which each of the eight subunits is an essential protein in yeast. If the GCP components are not redundant, then what different functional properties might they possess? There are at least two large-scale interactions in which the γ-tubulin complex must take part. One is with the microtubule, which is likely to involve interaction of the γ-tubulin/GCP2/GCP3 subunits with tubulin heterodimers (see below) in a still ill- defined way. The other interaction is with the centrosome, or its equivalent in other organisms. The relatively low level of conservation of GCP4, GCP5, and GCP6 among eukaryotes suggests that they might be involved in this interaction; the organelles that carry out microtubule organization in eukaryotes are morphologically divergent, and thus the interacting proteins might be different.
The stoichiometry of the components within the γ-tubulin complex also suggests distinct functions for the individual components. Previous studies have assessed the stoichiometry of the components in the γ-tubulin complex by protein quantification, either by dye-binding in gels (Zheng et al., 1995
; Oegema et al., 1999
) or by immunoreactivity on Western blots (Fava et al., 1999
). These studies revealed that γ-tubulin, GCP2, and GCP3 are the most abundant members of the complex. We took a complementary approach to determine whether individual components are present in single or multiple copies within the complex, relying on the ability to distinguish tagged and endogenous proteins in immunoprecipitates. Electron microscopy of the Drosophila
γ-tubulin complex has shown that it consists of a ring of repeated subunits (Oegema et al., 1999
), topped with an asymmetrical cap (Moritz et al., 2000
). Based on the observations that γ-tubulin, GCP2, and GCP3 form a discrete subcomplex in yeast and Drosophila
and that these proteins are the most abundant in the complex, it has been proposed that this γ-tubulin/GCP2/GCP3 subcomplex comprises the repeated subunit of the ring (Knop et al., 1997
; Oegema et al., 1999
). Consistent with this, we find that these three components are present in multiple copies (this study; Murphy et al., 1998
). GCP5 is present in a single copy per complex, indicating that it must not be part of a repeated structure and is thus likely to be in the cap. GCP4, however, is present in multiple copies. If GCP4 resides in the cap, then it might interact with the repeated ring subunits. It is also possible that GCP4 resides within the repeated ring subunits themselves, although this seems less likely based on its absence from yeast.
The γ-tubulin complex is present in the cytoplasm and at the centrosome and is thought to be active for nucleation only at the centrosome in vivo. The function of the cytoplasmic γ-tubulin complex is not known, but one possibility is that the cytoplasmic form is a free pool that can be recruited to the centrosome to increase microtubule nucleation when needed, such as at mitosis. What difference(s), if any, exist between the cytoplasmic and centrosomal forms of the γ-tubulin complex? We have identified six proteins in the cytoplasmic form of the human γ-tubulin complex. Each of these proteins is also present at the centrosome and interacts with microtubules, presumably as part of the larger complex. Thus, the cytoplasmic form does not have any known subunits that are lost upon either interaction. Further characterization of the cytoplasmic and centrosomal forms will allow a determination of whether they differ in more subtle ways, such as modification of any of the constituent proteins.