Functional Dissection of the Drosophila melanogaster Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I

摘要

The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo . Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila . Author Summary The accurate duplication and segregation of chromosomes during cell divisions are prerequisites for ensuring genetic stability within an individual organism and in entire populations. Among the many components involved in regulating these processes, a protein complex called condensin plays a crucial role in shaping mitotic chromosomes, so that they can be faithfully distributed. Many organisms contain two of these condensin complexes (condensin I and II), which both have been shown to be required for accurate chromosome distribution. In the fly Drosophila melanogaster , condensin II appears to lack one of its components, called Cap-G2. We have tested the hypothesis whether the corresponding component of condensin I (Cap-G) might also participate in the assembly of condensin II. Careful analyses of complexes formed in the living organism or in the test tube argue against Cap-G being part of condensin II. Moreover, our results question the very existence of a soluble condensin II complex in flies, as opposed to other organisms. Surprisingly, a substantially truncated variant of the essential Cap-G still supports development of living and fertile flies. As this variant localizes within the cell differently from full-length Cap-G, our results show that localization of a protein does not always determine its function.