How bacteria sort their lipoproteins (Lol!)

Bacterial lipoproteins are proteins with covalently-attached lipid molecules that anchor the protein to the cytoplasmic or outer membrane.  The lipid molecules are attached to the cysteine located at the amino terminus of the lipoprotein.  The lipoprotein's protein component, being hydrophilic (water-loving), sticks out from the membrane. Different bacterial lipoproteins participate in a variety of functions, including transport of molecules, stabilization of the cell wall, signal transduction, motility, and interaction with host molecules.

The Lyme disease spirochete Borrelia burgdorferi is exceptional in that a number of different lipoproteins have been found on its surface.  Most other bacteria lack (or have few) surface-exposed lipoproteins.  To give one example, the figure below shows the arrangement of lipoproteins in the cell envelope of E. coli, home to roughly 90 lipoproteins, none known to be displayed on the surface.  Lipoproteins are depicted as colored ovals with the attached squiggles representing the lipid molecules. To perform their functions properly, some lipoproteins must be anchored to the outer leaflet of the inner membrane (blue ovals) whereas the rest must be anchored to the inner leaflet of the outer membrane (red ovals).  In both cases, the protein component of the lipoprotein protrudes into the periplasm.  The figure also shows the other major category of membrane proteins, the integral membrane proteins, which are embedded in the membrane.  There are also proteins that reside in the periplasm, which are not depicted in the figure.

Figure 1 of Narita and Tokuda (2010)

In this post I will describe how lipoproteins are brought to their correct location in the bacterial envelope.  I will first describe how lipoproteins are sorted in E. coli since that's where most of the earlier work was conducted. Since many other diderms (bacteria having two membranes) have homologs of the proteins used by E. coli to export and sort lipoproteins, E. coli is a good model for studying localization of lipoproteins.  Monoderm bacteria also have lipoproteins, but since they have only one membrane, they don't need to worry about sorting lipoproteins.  (I will save the explanation of how lipoproteins get to the bacterial surface for a future post.)

Most proteins to be exported out of the cytoplasm are marked with an amino-terminal signal peptide ≈20 amino acids in length.  The sequences of the signal peptides (plus five additional amino acid residues) from two E. coli lipoproteins are shown below.  A cytoplasmic membrane protein complex called the Sec translocon transfers proteins harboring the signal peptide to the periplasm, where the signal peptide is lopped off by one of two signal peptidases.  Signal peptidase I cleaves off the signal peptide from nonlipoproteins (such as periplasmic or transmembrane outer membrane proteins), and signal peptidase II slices off the signal peptide from lipoproteins.

All lipoproteins harbor a short sequence called a "lipobox" at the end of the signal peptide (underlined in sequences below).  The lipobox consensus sequence  is -(leu, ala, val)_-4-leu_-3-(ala, ser)_-2-(gly, ala)_-1↓cys₊₁, with the arrow specifying the cleavage site for signal peptidase II and the subscripts denoting positions relative to the cleavage site.

E. coli Braun's lipoprotein (OM) MKATKLVLGAVILGSTLLAG↓CSSNA...

E. coli lpp-28 (IM)           MKLTTHHLRTGAALLLAGILLAG↓CDQSS...

(IM, inner membrane; OM, outer membrane)

The lipobox is recognized by the inner membrane enzyme phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt).  Before the signal peptide is removed, Lgt attaches diacylglycerol to the sulfhydryl (-SH) of the lipobox cysteine.  After the signal peptide is cleaved off by signal peptidase II, another inner membrane enzyme, apolipoprotein N-acyltransferase (Lnt), attaches a fatty acid molecule to the newly exposed amino (-NH₃) group of the cysteine.  Only exported proteins with lipoboxes become lipidated.  The lipoprotein remains associated with the inner membrane throughout these processing steps.

The machinery responsible for sorting lipoproteins to the outer membrane is the LolCDE protein complex, a type of ABC transporter that sits in the inner membrane.  Lol stands for lipoprotein outer membrane localization.  LolCDE recognizes the lipidated cysteine at the amino terminus of lipoproteins.  LolCDE loads lipoproteins onto the periplasmic protein LolA, which ferries lipoproteins to the LolB receptor, a lipoprotein that protrudes from the periplasmic face of the outer membrane.  After capturing the lipoprotein from LolA, LolB anchors the lipoprotein into the periplasmic layer of the outer membrane.

from figure 3 of Tokuda and Matsuyama (2004)

How does LolCDE know which lipoproteins are supposed to be delivered to the outer membrane and which need to stranded in the inner membrane?  For E. coli and other members of the Enterobacteriaceae family of bacteria, the answer is fairly simple.  Lipoproteins with aspartate at the +2 position (which follows the lipidated cysteine) remain in the inner membrane.

How does the +2 aspartate prevent transfer of lipoproteins to the outer membrane?  It turns out that LolCDE doesn't directly sense the amino acid at the +2 position.  Instead, the abundant membrane phospholipid phosphatidylethanolamine (PE) is thought to interfere with LolCDE recognition of the lipidated cysteine when asparatate is at the +2 position.  When the side chain carboxyl group (-COO^-) of the +2 aspartate interacts electrostatically with the positively-charged head group of PE, the fatty acids of PE become perfectly positioned to form hydrogen bonds with the lipid molecules attached to the cysteine (see figure below).  LolCDE is unable to recognize the amino-terminal cysteine associated with five fatty acid groups (three covalently bound to the cysteine and two from PE).  Thus asparatate, when it follows the cysteine, acts indirectly as a Lol avoidance signal.  The amino acid at the +3 position can also influence the Lol avoidance signal.  For example, negatively-charged amino acids (aspartate and glutamate) at the +3 position strengthen the +2 aspartate Lol avoidance signal by stabilizing the complex between phosphotidylethanolamine and the +2 aspartate (see figure below).

Modified from figure 6 of Tokuda and Matsuyama (2004)

Additional studies with engineered lipoproteins have shown that phenylalanine, tryptophan, tyrosine, lysine, and proline, although rarely found in lipoproteins at the +2 position, can also serve as inner membrane retention signals when asparagine is at the +3 position.  Since none of these are negatively-charged amino acids, the mechanism for avoiding LolCDE must differ from those lipoproteins having aspartate at the +2 position.

The nature of the sorting signal differs for bacteria that are not members of Enterobacteriaceae.  For example, the three amino acids at positions +2 through +4 dictate whether lipoproteins will remain in the inner membrane of Pseudomonas aeruginosa.  For the spirochete B. burgdorferi, a clear rule has yet to emerge from the few studies that have been done.  What can be said is that negatively-charged amino acids (aspartate and glutamate) placed within the first several amino acids following the lipidated cysteine sometimes allows the lipoprotein to remain in the membrane.  Whether the negatively-charged amino acid functions as an inner membrane retention signal depends on which amino acids are surrounding it.  It is not yet possible to simply look at the amino-terminal sequence of B. burgdorferi lipoproteins and confidently predict in which membrane they will be found.

Although the "+2/+3/+4 rule" is useful for predicting whether a newly discovered lipoprotein will be found in the inner or outer membrane, it may not give the complete picture of all of a lipoprotein's features that govern its localization.  The rules for sorting lipoproteins were worked out primarily by examining the localization of engineered fusion proteins consisting of the amino termini of lipoproteins (signal peptide with lipobox plus the first several amino acids following the lipobox cysteine) fused to unrelated reporter proteins such as red fluorescent protein (RFP) from corals.  For example, placing asp at the +2 position of such a fusion protein would cause RFP to be retained in the inner membrane.  Changing the +2 amino acid to serine would cause RFP to be transported to the outer membrane.  However, localization of a full-length lipoprotein may not be altered by simply changing its +2 amino acid from aspartate to another amino acid or vice versa.  This indicates that the rest of the lipoprotein, the part that's removed when reporters are used, also influences the localization of lipoproteins.


References

TOKUDA, H. (2009). Biogenesis of outer membranes in Gram-negative bacteria. Bioscience, Biotechnology, and Biochemistry, 73 (3), 465-473 DOI: 10.1271/bbb.80778

TOKUDA, H. (2004). Sorting of lipoproteins to the outer membrane in E. coli. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1693 (1), 5-13 DOI: 10.1016/j.bbamcr.2004.02.005

Schulze, R., & Zückert, W. (2006). Borrelia burgdorferi lipoproteins are secreted to the outer surface by default. Molecular Microbiology, 59 (5), 1473-1484 DOI: 10.1111/j.1365-2958.2006.05039.x