ZFYVE9C (Otherwise known as SARA) is a scaffold protein involved in the association of various protein components as part of the TGFB signalling pathway. The equivalent in the BMP signalling pathway is a protein known as Endofin so studying SARA (which is slightly easier) might give us useful insights for both proteins. For more details on this project please see this blog post.
One of the unanswered questions about SARA is what does one of the major domains that it has actually do? (The domain is known as a DUF domain, or a Domain of Unknown Function!) We know the structure but that hasn’t given us any clues as to its function. One thought was that it might be involved in recruiting type I or type II receptors close to their binding partners such as one of the SMADs to allow signal propagation.
I purified the DUF domain of SARA and mixed it with either excess TGFBR1 or excess TGFBR2 (the two relevant receptors in the TGFB pathway). I tested the receptors in both the unphosphorylated (inactive) and phosphorylated (active) states. After incubating the samples for 30 minutes while concentrating the volume back down to <5ml, I ran them down a gel filtration column to see if the two proteins bound together sufficiently to shift the peaks coming off the column which would indicate they were acting as a complex of larger mass.
Overlay of the UV traces from all the size exclusion gel filtration runs to show the relative elution volumes of the peaks confirming no shift in the ZFYVE9C peak consistent with no complex being formed.
Unfortunately I didn’t see any shift in the peak for SARA suggesting that either the two proteins don’t bind or that the affinity is weak enough that they fall apart under the conditions of the gel filtration column.
The next thing to try will be to purify the proteins again but not to cleave the tag on one of the binding partners (either the receptors or the SARA protein) and try to do a pull down experiment to test whether this shows any association or not.
You can find more details over at Zenodo.