A New ABC-Transporter Mediating the Detachment of Lipid-modified Proteins from MebranesYakushi, et al. 2000 Nat Cell Bio
Signficance
The cell membrane has many functions: regulating the passage of solutes in and out of the cell, facilitating cell-cell communication and interaction with the external millieu, defining cell size and shape, and even providing a surface for mediating reactions. Many of these functions are carried out by membrane proteins, but how do they get there? This can get even more complicated, because as you know, gram-negative bacteria (e.g. E. coli) have two plasma membranes...
These researchers identified the protein complex that's responsible for detaching lipoproteins from the inner membrane and bringing them to the outer membrane. This sounds like an arduous task, for these fat-soluble proteins have to cross the polar periplasm before arriving at the nonpolar outer membrane.
The type of transporter that they found is called an ABC transporter. This superfamily is responsible for using ATP energy to transport all different things across membranes, from small molecules and drugs to big proteins, and so have an interesting mix of conserved motifs along with highly variable pore sites. E. coli has around 57 in total (10 subfamilies) - 44 have associated partners and are thought to be involved in solute uptake, and the rest don't have partners, and are thought to be responsible for solute export. But LolCDE don't export - they just detach! And they analyzed sequences to find homologues in other species too, making this a conserved system in gram-negative bacteria.
There are over 80 E. coli lipoproteins, and 90% of them are on the outer membrane. I think many of them may be virulence factors too.
Background
Lipoproteins are transported across the inner membrane via the Sec translocon and a signal sequence. Once there, an N-terminal Cys gets aminoacylated with the lipid component.
LolA is a water-soluble chaperone in the periplasm, that helps to bring lipoproteins across the polar periplasmic space and into the outer membrane. If you don't have LolA, then proteins get stuck in the inner membrane.
LolB is a receptor for the LolA-lipoprotein complex, and basically accepts the lipoprotein for incorporation into the outer membrane.
But! How do proteins get from the inner membrane to LolA?...
Results
I thought they set up a pretty cool assay- they reconstructed proteoliposomes (from solubilized inner-membrane proteins + LolA + outer-membrane lipoprotein Pal + ATP + phospholipids) as basically chunks of membrane, and tested whether lipoproteins were getting transported or not, by analyzing supernatant (soluble) and pellet (insoluble, liposome-bound) fractions with PAGE. Is this an accurate reconstitution of the system? They seem to think so, but I would've liked to see them make it work for a few more candidate lipoproteins besides Pal.
Using classical chromatography, they then figured out what inner-membrane proteins in the mix were responsible for relasing the lipoprotein to LolA. They cloned the gene cluster for these proteins, purified them (LolCDE), and determined their ratios in a complex together.
By comparing the sequence to other ABC transporters, they identified several residues to mutate and test. The mutants inhibited the growth of cells that were Lpp+, but not Lpp- cells (Lpp, when mislocated, binds to peptidoglycan and inhibits cell growth).
They did another cool thing I didn't know about, which was create spheroblasts out of the growth-inhibited cells. You basically inhibit bacterial cell division and form long strings - you can then digest the cell wall, and they turn into big spheres! They found that the mutant LolD prevented proteins from getting released from the spheroplast, even with LolA added.
They also created a mutant lacking LolCDE that couldn't grow, and cells deficient in LolCDE couldn't release lipoproteins. After that, they used proteinase K digestion of the spheroplasts to prove that Pal wasn't translocated, but rather it was detached (completely digested)
Identification of Two Inner-membrane Proteins Required for the Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli
Ruiz, et al. PNAS 2008
Significance
Lipopolysaccharides are, for many bacteria, an essential membrane component, consisting of sugar+fat together in one molecule. The last article talked about Lol's involved in transporting lipoproteins across the periplasm. Lipopolysaccharides, on the other hand, have the Lpt system which also posseses an ABC transporter. They had found LptB, the ATP-binding domain, but couldn't find the transmembrane part until now - LptF and LptG.
Results
How do you find a mystery protein? That's a hard question, and there are different approaches, but usually you screen knockout/deletion mutants, until you find a key phenotype. This group, however, used bioinformatics. They knew the missing transporter must be conserved, because it's essential in gram-negative endosymbionts. What helps is that this family of bacteria lose a lot of their nonessential genes when evolving with their hosts (ah, connection to last entry!).
When probing other species, they found linkage of LptF and LptG to other Lpt genes, and even regions of gene overlap. They also found that deletion mutants were inviable. Mutants that only express low levels are sensitive to hydrophobic antibiotics, signifying a defective outer membrane. These low-level mutants were also found to make defective lipopolysaccharide, as determined by gel electrophoresis. They used an additional waaL knockout to show that LptFG must be acting after lipopolysaccharide export by MsbA (WaaL is involved in assembling lipopolysaccharide O-antigen...lipopolysaccharides are composed of lipid A, core, and O-antigen).
The next step was to demonstrate that LptFG weren't acting on outer membrane proteins. Apparently, when defects in assembly of outer membrane proteins occurs, they get degraded by the periplasmic protease DegP. But LptFG depletion strains all had normal levels of outer membrane proteins (they used OmpA and LptD).
When there are defects in lipopolysaccharide biogenesis, phospholiipids get translocated to the outer leaflet of the outer membrane. This activates PagP, which palmitoylates lipid A (adds a seventh lipid). They detected this increase in the weight of lipid A using MALDI-TOF mass spec for both LptF and LptG depletion mutants.
Finally, they use a pulse-chase experiment to determine whether, when they stop LptFG induction, de novo synthesized lipopolysaccharide was palmitoylated or not. Using TLC (with not good enough resolution, in my opinion), they did not see modified lipopolysaccharide in the outer membrane, suggesting that it never got there (defect in transport from the inner membrane).
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