Functional dissection of the APC
The APC is an unusually complex ubiquitin ligase consisting of at least 11 evolutionary conserved subunits. In contrast, several ligases are known to function as monomeric enzymes. To understand the function of the APC we intend to define the functions of individual subunits. One of APC’s subunits (Apc11) consists of a RING finger, which has been recognized in several proteins suspected to function in the ubiquitin system. To test whether Apc11 might constitute APC’s catalytic centre, we established an in vitro system consisting of purified components expressed in bacteria. We found that Apc11 by itself has the properties of a minimal ubiquitin ligase. It efficiently ubiquitinates cyclins but does not require the destruction box, a motif essential for recognition of APC substrates by the holoenzyme in vivo.

In the future, we plan to compare the properties of Apc11 with that of the complex to learn what functions are contributed by other subunits.

We next tried to reconstitute Apc11-containing subcomplexes from by co-expressing several subunits in bacteria. Although we could produce subcomplexes, the other subunits did not alter the activity of Apc11.

Rather than reconstituting the APC, breaking down the complex into functional sub-complexes might be another strategy. This approach was initiated by our discovery of two novel subunits in large-scale purifications of the APC from yeast. One of these is called Swm1/Apc13 and was thought to be a protein unique to budding yeast. However, bio-informatics analysis by Bianca Habermann in the MPI-CBG suggested related protein in most if not all eukaryotes. Indeed, we could detect the predicted homologs in the APC from fission yeast and from human cells. Analysis of mutants revealed that Swm1/Apc13 is required for the efficient degradation of APC substrates and the timely execution of APC-dependent cell cycle events. In cells lacking Swm1/Apc13 the APC dissociates into two sub-complexes. The work on Swm1/Apc13 has just been submitted for publication. In the future, we plan to use mutations in SWM1 and other subunits to “split” the APC into sub-complexes and analyse their functions.

Generation of the APC-Cdc20 holo-enzyme
The APC only functions as an ubiquitin ligase after binding to one of several activator proteins of the Cdc20 family. The APC-Cdc20 holo-enzyme is essential for entry into and exit from anaphase. To analyse the formation of the APC-Cdc20 complex, we purified the activator and found that most of Cdc20 is not associated with the APC but with the CCT chaperonin, which was thought to be a folding machine specific for actin and tubulin. We found that the CCT is required for Cdc20’s ability to bind and activate the APC. In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis. The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates APC during G1. Since Cdc20 was associated with the CCT also in human cells, we propose that folding of the Cdc20 family of APC activators is an essential and evolutionary conserved function of the CCT chaperonin. This work was published in the July issue of Molecular Cell.

Standing of the research project in national and international comparison
The lab has acquired experience in the identification and analysis of novel proteins and interactions using a combination of biochemical and molecular genetics methods in yeast. This approach is further strengthened by collaborations with other MPI-CBG groups, especially with mass spectrometry and bioinfomatics. Within the MPI-CBG, the lab is therefore in a good position to dissect the function of a protein complex as complicated as the APC. Very high levels of proteases and the poor synchrony have so far discouraged the use of biochemical approaches to study the meiotic cell cycle in yeast. The development in the lab of techniques for the large-scale purification of protein complexes including the APC from synchronized meiotic cultures therefore provides a considerable advance.