Final Report Summary - RZZ (Structural and functional characterization of the RZZ Complex)
This project was concerned with the structural and functional characterisation of the RZZ protein complex (800-kDa) that is named after the initials of its components, Rod, Zwilch and Zw10 (1). The RZZ is implicated in the execution of mitosis, where it is recruited to kinetochores (KTs) via an interaction with Zwint-1. The RZZ acts to recruit dynein (2) as well as the spindle assembly checkpoint (SAC) proteins Mad1 and Mad2 (3). Once proper attachment is achieved it also removes the SAC proteins from the kinetochore. Thus, the RZZ lies at the crossroad between the KT-MT attachment process and the SAC. How the RZZ fulfils these different roles and how it integrates signals from the attachment machinery was addressed in this study.
A number of results of this project have been published (4) and show that the structural analysis of the RZZ complex reveals a common ancestry with multisubunit vesicle tethering machinery. We applied structural analysis, in silico predictions, and biochemical and cellular approaches to investigate the structure and topology of the three RZZ subunits. The X-ray structure of human Zwilch, the first for an RZZ subunit, reveals a novel fold. We also identified neuroblastoma-amplified gene (NAG) as a Zw10 binder. We show that Rod and NAG are homologous proteins whose architecture, an N-terminal beta-propeller followed by a alpha-solenoid domain, is characteristic of subsets of nucleoporins and vesicle coat subunits. Rod and NAG participate in distinct complexes of Zw10, the RZZ and the NRZ. The latter is related to the Dsl1 complex of S. cerevisiae, a multisubunit tethering complex required for retrograde trafficking of COPI vesicles from the Golgi to the ER (5). Thus, the RZZ complex, which is limited to metazoans, might have evolved from the evolutionary conserved NRZ / Dsl1 complex, exploiting the dynein-binding capacity of ZW10 to direct dynein to metazoan KTs.
A major effort has been put into obtaining full length RZZ complex. After many failed attempts at expressing Rod, we have finally managed to express Rod in insect cells. In parallel the native complex was purifed from human cells using a stable cell line that expressed GFP-tagged Zwilch. We can now get a pure sample and are ready to scale up the process to purify enough material for subunit mapping and electron microscopy. The isolation of the RZZ complex was a major challenge and a crucial milestone of this project and this goal has now largely been achieved. This now permits to carry out a broad range of biochemical and structural analyses that will address the remaining points of this proposal.
Another aim was to get some insights into how the RZZ interacts with other KT proteins and how this function could be regulated. Interaction studies between Zwint-1, Zw10 and Zwilch have revealed that these two proteins alone are not sufficient for interaction. We will now test if the full-length complex can directly interact with Zwint-1 or whether some posttranslational modifications are required. We carried out cell biological essays to inhibit mitotic kinases, and the interference of some of these abolishes the recruitment of the RZZ to the KT but does not affect the localisation of its receptor Zwint-1. This strongly suggests a requirement for phosphorylation for the interaction. In parallel, we are trying to map the phosphosites on native RZZ proteins and preliminary data are now available that will most likely allow us to map the relevant sites for phosphorylation in the near future.
The objectives set in the proposed project have been partially achieved and new lines of research were pursued in order to lead to a first publication of the project. The project has also opened new lines of research that are now being actively pursued and should lead to one or more publications in the near future.
The socio-economic impact has two main implications: first a contribution towards a better understanding of the molecular mechanisms of mitosis, essential in targeting malfunctioning cell behaviour that results in uncontrolled cell division and cancer. Second, the training received has been invaluable from a scientific point of view and will contribute towards an independent research career.
1. Karess R. (2005) Trends Cell Biol. 15: 386-392
2. Starr D. A., Williams B. C., Hays T. S., Goldberg M. L. (1998) J. Cell Biol. 142: 763-774
3. Buffin E., Lefebvre C., Huang J., Gagou M. E., Karess R. E. (2005) Curr. Biol. 15: 856-861
4. Civril* F., Wehenkel* A., Giorgi F. M., Santaguida S., Di Fonzo A., Grigorean G., Ciccarelli F. D., Musacchio A. (2010) Structure 18: 616-26 *equal contribution
5. Schmitt H. D. (2010) Trends Cell Biol. 20: 257-68
A number of results of this project have been published (4) and show that the structural analysis of the RZZ complex reveals a common ancestry with multisubunit vesicle tethering machinery. We applied structural analysis, in silico predictions, and biochemical and cellular approaches to investigate the structure and topology of the three RZZ subunits. The X-ray structure of human Zwilch, the first for an RZZ subunit, reveals a novel fold. We also identified neuroblastoma-amplified gene (NAG) as a Zw10 binder. We show that Rod and NAG are homologous proteins whose architecture, an N-terminal beta-propeller followed by a alpha-solenoid domain, is characteristic of subsets of nucleoporins and vesicle coat subunits. Rod and NAG participate in distinct complexes of Zw10, the RZZ and the NRZ. The latter is related to the Dsl1 complex of S. cerevisiae, a multisubunit tethering complex required for retrograde trafficking of COPI vesicles from the Golgi to the ER (5). Thus, the RZZ complex, which is limited to metazoans, might have evolved from the evolutionary conserved NRZ / Dsl1 complex, exploiting the dynein-binding capacity of ZW10 to direct dynein to metazoan KTs.
A major effort has been put into obtaining full length RZZ complex. After many failed attempts at expressing Rod, we have finally managed to express Rod in insect cells. In parallel the native complex was purifed from human cells using a stable cell line that expressed GFP-tagged Zwilch. We can now get a pure sample and are ready to scale up the process to purify enough material for subunit mapping and electron microscopy. The isolation of the RZZ complex was a major challenge and a crucial milestone of this project and this goal has now largely been achieved. This now permits to carry out a broad range of biochemical and structural analyses that will address the remaining points of this proposal.
Another aim was to get some insights into how the RZZ interacts with other KT proteins and how this function could be regulated. Interaction studies between Zwint-1, Zw10 and Zwilch have revealed that these two proteins alone are not sufficient for interaction. We will now test if the full-length complex can directly interact with Zwint-1 or whether some posttranslational modifications are required. We carried out cell biological essays to inhibit mitotic kinases, and the interference of some of these abolishes the recruitment of the RZZ to the KT but does not affect the localisation of its receptor Zwint-1. This strongly suggests a requirement for phosphorylation for the interaction. In parallel, we are trying to map the phosphosites on native RZZ proteins and preliminary data are now available that will most likely allow us to map the relevant sites for phosphorylation in the near future.
The objectives set in the proposed project have been partially achieved and new lines of research were pursued in order to lead to a first publication of the project. The project has also opened new lines of research that are now being actively pursued and should lead to one or more publications in the near future.
The socio-economic impact has two main implications: first a contribution towards a better understanding of the molecular mechanisms of mitosis, essential in targeting malfunctioning cell behaviour that results in uncontrolled cell division and cancer. Second, the training received has been invaluable from a scientific point of view and will contribute towards an independent research career.
1. Karess R. (2005) Trends Cell Biol. 15: 386-392
2. Starr D. A., Williams B. C., Hays T. S., Goldberg M. L. (1998) J. Cell Biol. 142: 763-774
3. Buffin E., Lefebvre C., Huang J., Gagou M. E., Karess R. E. (2005) Curr. Biol. 15: 856-861
4. Civril* F., Wehenkel* A., Giorgi F. M., Santaguida S., Di Fonzo A., Grigorean G., Ciccarelli F. D., Musacchio A. (2010) Structure 18: 616-26 *equal contribution
5. Schmitt H. D. (2010) Trends Cell Biol. 20: 257-68