Methods for the investigation of protein-protein interactions (PPIs) in living cells1
are suboptimal for the analysis of dynamic and short-lived enzyme-substrate interactions. Therefore, data on kinase-substrate and phosphatase-substrate interactions are mostly absent from current PPI databases2
. To overcome some of the limitations we developed M-Track (for 'methyl-tracking'), an assay that uses an enzyme-catalyzed methylation of a specific substrate lysine for the detection of PPIs in yeast (). A biotinylation-based enzyme-substrate approach has been described for mammalian cells3
, but it is inappropriate for yeast studies because of their high biotinylation background. In addition, this approach appeared unsuitable for detection of short-lived PPIs (Supplementary Fig. 1
and Supplementary Note 1
). In M-Track, the bait protein is expressed as a fusion protein with the H320R mutant of the human histone lysine (K) methyltransferase (HKMT) SUV39H1, which possesses a more than 20-fold higher catalytic activity than the wild-type enzyme4
and, despite lacking the chromodomain for substrate recruitment, is sufficient for histone H3 Lys 9 (K9) trimethylation4
. The prey protein is a fusion protein with three or four tandemly arranged copies of amino acids 1–21 of histone H3 (H3 tag), followed by hemagglutinin (HA) epitope tags. As a readout system we used western blot analysis of whole-cell lysates, for which we generated monoclonal antibodies with high specificity for the different H3K9 methylation states (Supplementary Fig. 2
The M-Track assay for detection of stable and transient PPIs in the HOG pathway
To assess the performance of M-Track, we first analyzed the rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP12-rapamycin binding (FRB)5
. In the absence of rapamycin, we detected hardly any methylation of the prey FKBP-H3-HA (). Upon rapamycin addition, the methylation levels increased, with monomethylation peaking after 5 min and trimethylation increasing steadily between minutes 5 and 15. Next, we determined the influence of stress conditions on our assay system (Supplementary Fig. 3
). The methylation rates rose substantially with increasing temperatures, but they were not affected by osmotic or oxidative stress.
Because we were interested in the detection of stress-induced PPIs, we studied the high osmolarity glycerol response (HOG) pathway in which Sho1, a transmembrane protein, interacts stably via an SH3 domain with the MAPKK Pbs2 that gets activated by the upstream MAPKKK Ste116
. After determining that the tagged proteins are functional in vivo
(Supplementary Fig. 4
), we used M-Track to monitor binding to Pbs2 of Sho1-SH3 mutants known to have increasing dissociation constants for this interaction7
. We expressed H3-tagged Sho1 and HKMT-tagged Pbs2 in strains either lacking or expressing the endogenous proteins, which additionally lack signaling through the Sln1 branch (ssk2
Δ) (). In the sho1
Δ strain, the methylation signal decreased with increasing dissociation rates of the mutants and was undetectable for the ΔSH3 mutant allele (). In comparison with wild-type Sho1, we observed a 70% and 92% reduction in methylation signal for the Y8A and Y54M mutants of Sho1, respectively. Furthermore, we observed less Hog1 phosphorylation with the SH3 mutants, indicating that the results of the M-Track methylation assay correlated with biological outcome.
When endogenous untagged Sho1 and Pbs2 were present (), however, we could no longer observe the Kd
-dependent differences in methylation of Sho1 mutants, although wild-type Sho1 was still more strongly methylated than the mutants. Even the ΔSH3 mutant was methylated by HKMT-Pbs2 when endogenous Sho1 was present, an observation that can be explained by the assumed homo-oligomerization of Sho1 at the plasma membrane ()8
. We conclude that methylation levels in M-Track can reveal binding affinities but also that close proximity of proteins in a membrane or in a complex could generate a methylation signal. We could also use M-Track to detect the short-lived Ste11-Pbs2 interaction and to track the changes of this MAPKKK-MAPKK interaction in response to osmotic stress in vivo
, as indicated by a threefold increase in the methylation signal ().
Other short-lived interactions that are notoriously difficult to detect are the ones between protein serine/threonine phosphatases, such as PP2A, and their substrates. In yeast two regulatory B-subunits of PP2A, Cdc55 and Rts1, exert non-redundant functions, presumably by targeting distinct substrates9,10
, although the identities of these substrates have remained elusive. A probable PP2A–Cdc55 specific substrate is the nucleolar protein Net1 11
, but there is no evidence for direct interaction of these proteins12
. We used M-Track to investigate this question (). After testing that the fusion proteins were functional ( and Supplementary Fig. 5a,b
), we probed for an interaction between Myc-HKMT-Cdc55 and H3-HA-Net11-600
(Net1 amino acids 1-600) (). Despite their overexpression these proteins did not co-immunoprecipitate at substantial levels (Supplementary Fig. 6
). Upon galactose-induced bait expression, Myc-HKMT-Cdc55 reached a steady-state level after 2 h, whereas the prey level was constant over the entire time course. Concomitant with increasing Myc-HKMT-Cdc55 levels, we observed a time-shifted curve progression of the mono-, di- and trimethylated prey species. This indicated that the reaction mechanism is probably non-processive and suggested that Myc-HKMT-Cdc55 targeted H3-HA-Net11-600
several times, possibly at several sites.
M-Track detection of the short-lived PPI between PP2A-Cdc55 and its substrate Net1.
Consistent with this finding, Net1 is phosphorylated by cyclin-dependent kinase (Cdk) at several sites; these represent potential PP2A-Cdc55 targets13
. We conducted an M-Track assay with a Net1 mutant, 3Cdk, that lacks three of the mapped Cdk phosphorylation sites13
. Myc-HKMT-Cdc55 was unable to trimethylate the 3Cdk Net1 mutant (), indicating that efficient methylation depended on the presence of phosphorylatable Cdk sites. These results strongly suggest that Net1 is an in vivo
substrate of PP2A-Cdc55 holoenzymes. The HKMT domain alone or an HKMT fusion protein with the second regulatory subunit of PP2A, Rts1, showed very little trimethylation of H3-HA-Net11-600
(), in contrast to the results with Myc-HKMT-Cdc55. The inability of Rts1 to target Net1 was not due to a general impairment of its function by the HKMT fusion (Supplementary Fig. 5
). Moreover, we could detect an interaction between Myc-HKMT-Rts1 and its putative substrate Kin414
(Supplementary Fig. 7
). These findings indicated that M-Track detection of the hybrid protein interaction between PP2A and Net1 depended on the substrate specificity provided by the specific B-subunit rather than the HKMT.
For M-Track to efficiently detect fast catalytic PPIs, the methyltransferase reaction requires an HKMT mutant with high catalytic activity that also lacks the substrate recruitment domain, which reduces the HKMT's affinity for its endogenous substrate and minimizes false-positive results. Notably, the methylation level reflects the integrated sum of multiple transient or stable interaction events minus the protein turnover rate as demethylation is absent in yeast, which limits the use of M-Track the analysis of dissociation kinetics. Furthermore, the methylation rate will not only depend on the binding affinities and duration of protein interactions but also on steric parameters. The positions of the HKMT and H3 tags as well as the design and flexibility of the linkers may influence the readout of the system and must be empirically evaluated.
High-throughput discovery and phosphoproteome screens have identified large numbers of putative enzyme-substrate interactions that require validation by a method such as M-Track. The ability to discriminate between a direct and an indirect enzyme target will help determine the hierarchical structure of signaling cascades within existing data sets and will lead to a better understanding of signaling networks. In addition, the methylation signature left on proteins that have interacted can be used to define differences in biochemical profiles (for example, presence of post-translational modifications) between newly synthesized proteins or participants in different protein complexes. Moreover, the three-step enzymatic reaction catalyzed by the HKMT could be used as an indicator for PPI duration. The system may serve as a structural ruler within stable complexes by using differently sized spacers between the protein and the enzymatic domain. Finally, we envision several future developments of the system such as substrate identification screens using mass spectrometry or the quantitative analysis of dynamic PPI changes by microwestern arrays15