How Doa10p gets into the nucleus, or another freaky experiment done in yeast

I heard about this paper (Deng and Hochstrasser. Nature (06) 443:827-831) and took a look at it over the weekend. Wow! There are lots of goodies in there. And it showcases how manipulable yeast are. (As you can tell I am really jealous of researchers who use yeast as a model system.)

The premise of the paper is not bad either.

There had been some rumours that proteins could get degraded within the nucleus through the ubiquitin/proteosome pathway. Now to some this idea was heretical but this new paper gives some mechanistic info into how this process occurs.

Doa10p is an E3 ligase, that is it is the enzyme that attaches ubiquitin to the protein destined to be degraded. It's also a membrane bound ubiquitin ligase that is involved in the extraction of endoplasmic reticulum (ER) proteins that have a misfolded cytoplasmic domain (i.e. ERAD-C, see this note on ERAD and this note on the 3 types of ERAD). Previously Doa10p had also been implicated in the degradation of mat-alpha2 transcription factor, a nuclear protein involved in turning on genes in response to mating factor. So what's happening?

Doa10p had been localized to the peripheral ER but it was unclear if it diffused into the inner-nuclear membrane. Remember that the ER extends over the nucleus and is continuous with the outer-nuclear membrane (ONM) and inner-nuclear membrane (INM), however to get to the INM proteins have to pass through the nuclear pore complex (NPC). To degrade nuclear proteins, a part of Doa10p must thus pass through the NPC. To confirm this, Deng looked in cells that over express Nup53 and have an over abundance of INM (in these cells the INM looks wrinkled while the ONM is normal). Indeed Doa10p was enriched in the nucleus of these cells indicating that Doa10 does partition into the INM. How is Doa10p crossing the NPC to get to the INM? Well if you remember I wrote an entry on how the Blobel lab discovered that some INM proteins use a nuclear localization sequence (NLS) to cross the nuclear pore, so is this true for Doa10p? At first approximation it doesn't seem like Doa10p has an NLS. Now this may not be a problem, the nuclear pore complex only filters particles that are larger than ~25kDa, anything smaller can freely diffuse across. The authors first speculate that Doa10's cytoplasmic domain may be small enough to passively diffuse in, but if they make Doa10's cytoplamsic domain larger, it still can cross in. So what does it take to get in? Just like the Blobel paper, they screen cells that have mutant forms of each NPC component (called Nups) to figure out how the pore regulates the import of Doa10. Incredibly they find that Doa10 requires different Nups than what had been shown for other INM proteins! This would indicate that membrane proteins travel across the NPC via different mechanisms.

Then they perform a really crazy experiment. The authors want to prove that mat-alpha2 is indeed degraded by nuclear Doa10p. The problem is forcing Doa10p to remain out of the nucleus. To accomplish this feat the authors fuse Doa10p to coronin, an actin binding protein. The result is that Doa10p is retained in the cortical ER where it binds to actin filaments and thus sequestered away from the nucleus. Not surprisingly they find that these cells don't effectively degrade mat-alpha2. And then if cells are treated with actin depolymerizing drug (latrunculin), Doa10p is released from the peripheral ER, enters the nucleus and degrades Mat-alpha2.

Holly crap! Talk about showcasing the manipulability of yeast ... and also the idea of combining genetic manipulation with pharmacology.

So there you have it - the newest twist on how proteins reach the inner-nuclear membrane.

Ref:
Deng, M. and Hochstrasser M.
Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase.
Nature (06)443:827-831