Characterization of the LolA-LolB System as the General Lipoprotein Localization Mechanism of Escherichia coli

Abstract

The major outer membrane lipoprotein (Lpp) of Escherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane. We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins. All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA. Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent. This LolAindependent release was also strictly dependent on the outer membrane sorting signal. A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events. The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient. The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane. The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane. From these results, we conclude that lipoproteins in E. coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane.

Dominant negative mutant of a lipoprotein‐specific molecular chaperone, LolA, tightly associates with LolCDE

“…When LolA(F47E) was used in place of LolA, only a marginal amount of Lpp was released. In contrast to Lpp, a small amount of Pal was released in a LolA-independent manner and the addition of LolA caused the complete release of Pal as reported [6]. LolA(F47E) was found to be as active as wild-type LolA as to the release of Pal.…”

“…Taken together, these results indicate that LolA(F47E) is a leaky mutant defective in the release of lipoproteins. Since the release of Lpp more strongly depends on the LolA function than that of Pal [6], the defect of this mutant seemed to be more clearly observed in the release of Lpp.…”

“…The amounts of lipoproteins were determined with an ATTO Densitograph. Western blot analyses were performed as described [6]. The blots were densitometrically quantitated at least twice within 5% deviation.…”

“…As reported previously for LolA(R43L) [10], mutagenized lolA was expressed from a plasmid by the addition of arabinose, and defective LolA mutants that did not support the growth of TT016 in the absence of IPTG were isolated. Among these mutants, one carried on pAMF47E was found to inhibit the growth of TT016 not only in the absence (open circles) but also in the presence (closed circles) of IPTG, Spheroplasts were prepared from MC4100 cells harboring pTAN21 [6], which carried Ptac-pal, induced for 5 min with 1 mM IPTG. A: Pulse-labeling and the chase were performed as described under Section 2 in the presence of 10 Wg/ml LolA or LolA(F47E).…”

Characterization of the Pseudomonas aeruginosa Lol System as a Lipoprotein Sorting Mechanism

“…Purification of Lipoproteins-Wild-type Pal was overexpressed in JM83 carrying pTAN21 (15). Pal derivatives were expressed in JC7752 (⌬pal) (22) transformed with pTAN26 (15), pTAN21KS, pTAN21LI, or pTPH21 (23).…”