Hello everyone,
I am trying to decipher the interaction between different karyopherins (importin alpha and importin beta) and we also want to show them binding to an NLS containing cargo, to prove functionality.
So far, what I can see is binding of one specific importin beta to the different importin alphas we are investigating.
How I do this: I purify the karyopherins from Sf9 insect cells by IMAC, Ion exchange and size exclusion. They all are C-terminally tagged with GFP-6xHis. Additionally, we purified two different cargo constructs with NLS sequences on their C-terminus via MBP purification and size exclusion. Cargos are 14His-MBP-GFP-NLS(EGL13) and 14His-MBP-GFP-NLS(Myc). Then I simply mix different combinations of proteins (1uM each) in 300ul volume of buffered solution (HEPES pH 7.5), physiological salt concentration (150mM), 1mM DTT and 20mM Imidazole to ensure just binding of my bait (either alpha, beta or the cargo). I incubate the proteins with each other for 1 hour on ice and then simply pull down the bait via MagneHis beads. Binding is quite efficient and therefore facilitated for just 10 min. Then I take off the supernatant, elute with 1M Imidazole and see what comes along in the elution fraction and what stays in the supernatant. Whatever comes down with the bait, we assume is binding to the bait. Whatever comes down in the supernatant does not bind to the bait.
According to literature, binding of importin alpha to a cargo needs to occur in order to facilitate the binding of importin alpha to importin beta. The interaction of importin alpha with the cargo is crucial to liberate importin alpha from its auto-inhibitory state since this releases the importin beta binding domain (IBB) of importin alpha which otherwise forms intramolecular interactions with importin alpha itself and therefore inhibits binding to importin beta. Hence, importin alpha is able to bind importin beta when bound to a cargo.
So here is the problem: What I can observe is binding of one specific importin beta to all importin alphas we are investigating. However, in this scenario, no cargo is involved. If either the one or the other cargo construct (see above) is added, I see that this importin beta is binding importin alpha independent of importin alpha-cargo interaction since there is no cargo in the elution fraction (as quite obvious by the previous experiment). Even when the tags are cleaved off of the importins and I use the cargo as bait I see no co-elution of any other protein besides the bait (the cargo). Also, just incubation of the importin alpha constructs with the cargos shows no binding.
This is somewhat counter-intuitive that importin alpha binds importin beta without the requirement of a cargo. Additionally, we can't really explain why our importin alpha constructs are not binding any of those two cargos.
Is anyone experienced with in vitro work with karyopherins and their binding to each other in combination with an NLS containing cargo? Basically, we just want to see that the importin alpha constructs are functional by binding a cargo. Do we just have the wrong NLS-sequences on our cargo constructs? As far as I am aware they are canonical mono- and bi-partite NLS-Sequences. And lastly, why do our importin alphas bind to importin beta when there is no cargo around?
I would appreciate any experience, comment or suggestion you can share and I am looking forward to discussing this matter for anyone who is willing and interested.
Much thanks in advance!
Philipp
Hi,
I am wondering if you have any citation for this:
"According to literature, binding of importin alpha to a cargo needs to occur in order to facilitate the binding of importin alpha to importin beta."
Based on this article, it seems like Imp-a can bind to Imp-beta even when cargoes are not bound to Imp-a
https://www.cell.com.remotexs.ntu.edu.sg/trends/cell-biology/comments/S0962-8924(04)00195-3#secd41165973e406
Could you link this again differently please, I get directed to the university of Singapore when I click it. I’m curious.
I have to find it, but I’m sure I can send you some citation that states that imp-a auto-inhibits itself by binding to its importin beta binding domain unless it’s bound to a cargo. Will try to find it.
It would be great if you can send me some citations as I am looking for it too.
Check these papers for alpha binding to beta without cargoes:
1. Importin α: a multipurpose nuclear-transport receptor
2. Nucleocytoplasmic transport enters the atomic age (Fig. 2)
3. Characterization of the Importin-β binding domain in nuclear import receptor KPNA7
4. Quantitative analysis of nuclear localization signal (NLS)-importin alpha interaction through fluorescence depolarization. Evidence for auto-inhibitory regulation of NLS binding (This might be more relevant to what you are doing)
This is one paper that I quickly found: Article The Auto-inhibitory Function of Importin Is Essentialin Vivo
I would need to search a bit more if you want more citations since I do not have a lot of papers saved unfortunately.
Hey!
So I am reading a bit now and I think I might know how things might work with importin beta, alpha and the cargo and I think this is what you were trying to say. Please let me know what you think:
So what I found is that dissociation constants are somewhat high between a full length importin alpha and an NLS containing cargo. This is, I assume, due to importin alphas auto-inhibitory mechanism involving the IBB (importin beta binding) domain. However, when alpha binds to importin beta, alpha has increased affinity for NLS containing cargos since the IBB domain doesnt block NLS binding domains in this case. So this would mean that alpha binding to beta primes the alpha to bind an NLS.
The way I thought it works is the other way around. I assumed that alpha binds the NLS first, this sets free the IBB domain and therefore it can bind to importin beta.
I would really appreciate your opinion on this.
All the best :)
Hi,
After reading some papers, I think the model is as such:
Imp-a, in its auto-inhibition form, binds NLS with low affinity. The binding of NLS to Imp-a, in turns, allows Imp-b1 to bind to the IBB domain. Due to the binding of Imp-a/b1, it further enhances the NLS binding to Imp-a.
However, this is just a model. I could not find solid evidence showing Imp-b1 not binding to Imp-a without cargo binding. Also, I think there are a few papers that discuss the limitations of in vitro IP. Could the autoinhibitory structure be slightly different in vitro vs in vivo?
Well, I think what you described aligns pretty well with what I think too. If you look at dissociation constants (Kd) then Imp-a shows micro molar Kds for NLS cargos. However, if Imp-b is present the affinity increases usually more than 10-20-fold to Kds in the range of nano molar (at least in the papers I looked over quickly yesterday). Meaning that Imp-a in general has weak affinity to NLS in absence of and not bound to Imp-b. Additionally, dissociation constants for Imp-a and Imp-b show stronger affinity in vitro (around 10nM) than Imp-a to NLS (in the uM range), supporting what we described.
However, there seem to be at least some NLSs that still bind somewhat "good" to Imp-a without Imp-b present, like the well known SV40 and Nucleoplasmin in the range of 200nM to 400nM (not particularly awesome but ok).
I was also asking myself about the differences in vitro and in vivo. I would assume that there are differences. I read once that at least the affinities differ in vivo compared to in vitro assays. However, I could not find the one paper I read this so I cant give you any citation unfortunately. When it comes to auto-inhibition, maybe there might also be differences in vivo versus in vitro. I cant really tell.
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