Hello,

How do you generate the proton gradient? Do you use external buffers with different pH than the one that you used to make the liposomes?

Specificity is definitely sensitive to experimental conditions. In this context see also the recent work of Bill (WF) DeGrado. He mentioned this also in his recent 3Dsig talk - don't recall if that was on published or yet-unpublished work.

 Maybe you have just to take into account the possibility that the integrity of your proteoliposome vesicles could be affected by the misfolding of your protein, by the uncorrect ratio between protein and lipid amount, or other experimental conditions. These could cause an apparent transport due to a passive crossing of the radiolabeled substrate. Good luck!

I guess if the fold is incorrect your protein will most likely precipitate or not work at all. In some cases people seem to have problems with contaminants. Maybe you have porin contamination from the yeast mitochondria outer membrane? 

Thank you for your answers.

Initially, the proteoliposomes inner buffer is the same as the inner buffer, both pH7. Change of the outer pH is induced by addition of acidic/alkaline sucrose master-mix to create a final pH-difference of +1/-1/-2.

@Susana L A Andrade

Contamination with mitochondrial porins is possible in the experiment in question. Though yeast mitochondria shouldn't contain sucrose-porins, I guess? Maybe a relatively unspecific porin that get's co-purified by Ni-NTA? (Indeed, the "purified" eluate shows lots of protein bands that are definitively not the POI).

For this experiment I purified the mitochondrial membrane fraction as well, because the Western blots of previous purifications showed a strong presence of my POI in there, even if it shouldn't be that way (no signal peptide). Additional purification of the mitochondrial membrane fraction resulted in 55-fold higher total protein yield (elution from Ni-NTA) than compared to microsomal fraction only, POI gain was proportional.

Potentially wrong folding could lead to everything nonspecific, "transport against the proton gradient" might be interesting depending on ion/molecule transported and in/out concentration for the ion/molecule. E.g. single amino acid substitution leads to nonspecific transport, one of numerous examples http://journal.frontiersin.org/article/10.3389/fpls.2014.00600/abstract

How do you know the protein is misfolded? Removal of a signal peptide does not necessarily cause misfolding, rather mislocalization, no? Can you purify and reconstitute your protein with and without the signal peptide?

If the protein is misfolded chances are that it would lose some activity and /or may lead to non-specific transport, only empirical evidence (such as the evidence you are accumulating) can answer these questions. Can you think of other experiments to asses 1. Protein misfolding 2. Non-specific trasnport?

Good luck!

Fabian

You can test whether your protein is misfolded by running a CD spectrum to estimate its content of secondary structures or  measure its size with a sedimentation, Gel filtration or EM.

Good luck

Zhiping

I think that the unspecific transport activity that you detect is due to "leaky" liposomes. To confirm this, you should prepare your liposomes the usual way you do, without adding your transporter. I am afraid that if the protein is not folded correctly, it is unlikely to transport anything.