I am interested in detecting inhibition of lipoprotein transport to the outer membrane of E. coli. In several papers (Cock, 2005, JBC), I have noticed that authors created spheroplasts, radiolabeled to track newly formed protein, and then simply centrifuged their samples to separate spheroplasts from the media. They showed that radiolabeled lipoproteins remained in the spheroplast fraction unless they added lolA (periplasmic lipoprotein transporter), in which case the lipoprotein clearly moved to the supernatant fraction. It seems to me that spheroplast formation and simple centrifugation effectively separated the inner and outer membranes (into spheroplast and supernatant fractions). Why then do so many papers follow up with or solely rely on sucrose gradient centrifugation to separate the inner and outer membranes? This technique is more difficult and labor-intensive. Why not rely solely on spheroplasting to track protein localization to the inner or outer membrane?
The outer membrane is destroyed in the process of preparing spheroplasts, which is why the lolA protein causes the transported proteins to end up in the supernatant. Sucrose gradient centrifugation can be used to prepare separate fractions consisting of inner membrane and outer membrane.
There are different protocols for spheroplasting. Tokuda's protocol involve EDTA which disperses LPS and for this reason destroys the outer membrane. But the proteins released to the supernatant would also include a periplasmic fraction, so this method can't fully differentiate protein localization. So choosing the method depends on the experiment and the purpose, really. Spheroplasting is simple and can be easily used for screens, which Tokuda used to identify Lol proteins.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology