I have been studying a protein that has an LPXTG motif, so it would be expected to be sorted to the cell wall.

However, it only appears in the membrane fraction when I do a western blot. Does the presence of an LPXTG motif strictly dictate cell anchoring

or have proteins with LPXTG motifs been known to localise to other parts of the cell?

Well, if it really functions as a an LPXTG motif, one would expect some signal in the cell wall. It is possible that it has the peptide sequence, but it is not really used as a signal (less conserved areas near the motif also pertain to function), or for some reason the sortase system does not act on it (e.g. due to some sort of posttranslational regulation, if the signal in the membrane fraction is strong).

 

If the detection of the LPXTG motif is the only indicator that it is cell wall associated, my guest would be the first point. I remember a long time back a bioinformatician trying having problems to make sense out of predictions based on signal motifs. Especially the LPXTG motifs did not make any sense. The problem? He looked at a Gram negative bacterium ;p

Well, if it really functions as a an LPXTG motif, one would expect some signal in the cell wall. It is possible that it has the peptide sequence, but it is not really used as a signal (less conserved areas near the motif also pertain to function), or for some reason the sortase system does not act on it (e.g. due to some sort of posttranslational regulation, if the signal in the membrane fraction is strong).

 

If the detection of the LPXTG motif is the only indicator that it is cell wall associated, my guest would be the first point. I remember a long time back a bioinformatician trying having problems to make sense out of predictions based on signal motifs. Especially the LPXTG motifs did not make any sense. The problem? He looked at a Gram negative bacterium ;p

 

The protein is a Staphylococcal protein. I cloned the gene encoding it into another species of Staph. and in this host the protein was detected in the wall. This makes me think that my cell wall digest is insufficient (although I have observed other wall proteins with this protocol) or that the cell wall signals are not detected by the sortase in it's native Staphylococcal host but are in the other, maybe because it is cleaved in this host.

 

Thanks for your help.

Interesting. Sounds pretty conclusive that it is functional as a motif. I wonder if some different (regulatory) effects may play a role in the native host somehow. Did you happen to quantify the expression of the protein in the native vs the heterlogous expression?