Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin

Figure 2.38 Protein transport systems. (A) Cutaway view of the secretion sec channel. SecY, SecE, and SecG (not shown) form the translocase. SecY forms the channel, ring, and plug. The signal sequence of the transported protein moves the plug toward SecE. (B) Posttranslational secretion by the SecB-SecA system. SecB keeps the protein unfolded until it binds to SecA, which interacts with SecY. The signal sequence is removed, in this case by Lep protease. The exported protein is folded in the periplasm or may be secreted across the outer membrane by one of the dedicated secretion systems. (C) Cotranslational transport by the signal recognition particle (SRP) system. SRP binds to the first transmembrane domain as it emerges from the ribosome and then binds to the FtsY docking protein, bringing the ribosome to interact with SecY. The protein is translated, driving it into the SecYEG channel. The transmembrane domains of the protein somehow escape through the side of the channel into the membrane, in some cases with the help of the YidC protein, as shown.

      Besides forming the major part of the channel, a region of the SecY protein forms a hydrophobic “plug,” which opens only when a protein is passing through (Figure 2.38). The binding of a signal sequence (see below) in a protein to be transported causes the plug to move over toward SecE on the side of the channel, opening the channel. As a result, only proteins that have a bona fide signal sequence can be translocated.

The Signal Sequence

      As mentioned above, the defining feature of proteins that are to be transported into the inner membrane or beyond by the SecYEG channel is the presence at their N termini of a signal sequence. The nature and fate of this signal sequence depend upon the ultimate destination of the transported protein. For proteins that are to be transported through the inner membrane into the periplasm and beyond, the signal sequence is approximately 20 amino acids long and consists of a basic region at the N terminus, followed by a mostly hydrophobic region and then a region with some polar amino acids. In contrast, most proteins whose final destination is the inner membrane merely use their first N-terminal transmembrane domain as a signal sequence.

      If the protein is to be secreted through the membrane, the signal sequence is removed by a protease as the protein passes through the SecYEG channel (Figure 2.38B). The most prevalent of the proteases that clip off signal sequences in E. coli is the Lep protease (for leader peptide protease), but there is at least one other, more specialized protease called LspA, which removes the leader sequence from some lipoproteins destined for the outer membrane. Proteins that are destined to be transported beyond the inner membrane but have just been synthesized and so still retain their signal sequences are called presecretory proteins. When the short signal sequence is removed in the SecYEG channel, the presecretory protein becomes somewhat shorter before it reaches its final destination in the periplasm or the outer membrane or outside the cell.

The Targeting Factors

      The targeting factors recognize proteins to be transported into or through the inner membrane and help target them to the membrane. Which type of signal sequence a protein has determines which of the targeting factors directs it to the SecYEG translocon. Enteric bacteria like E. coli have at least two separate systems that target proteins to and through the membranes. The SecB system is dedicated to proteins that are directed through the inner membrane into the periplasm or exported from the cell. The signal recognition particle (SRP) system seems to be dedicated to proteins that are mostly destined to reside in the inner membrane. Another protein, SecA, participates in both pathways, at least for some proteins; it is found in all bacteria, but not in archaea or eukaryotes, although in eukaryotes other proteins may play a similar role.

      THE SecB PATHWAY

Proteins that have a removable signal sequence and are transported through the inner membrane into the periplasm or beyond are most often targeted by the SecB system in E. coli and the other bacteria that have it. The SecB protein is a specialized chaperone that binds to presecretory proteins either cotranslationally (e.g., as soon as the N-terminal region of the polypeptide emerges from the ribosome) or after they are completely synthesized, thereby preventing them from folding prematurely and ensuring that the signal sequence is exposed. The SecB chaperone passes the unfolded protein to SecA, which facilitates the association of the protein with the SecYEG channel, perhaps by binding simultaneously to the signal sequence and to a SecYEG heterotrimer (Figure 2.38B). Some bacteria have paralogs of SecA that may be dedicated to transporting only one or very few proteins. After SecA binds to the channel, the cleavage of ATP to ADP on SecA provides the energy to drive the protein into the channel, aided by the proton motive force of the membrane. As the protein passes through the channel, it loses its signal sequence, as shown in the figure. SecB is not an essential protein, and the cell can use DnaK or other general chaperones as substitutes for SecB to help transport some proteins.

      THE SRP PATHWAY

      The SRP pathway in bacteria generally targets proteins that are to remain in the inner membrane. It consists of a particle (the SRP) made up of both a small 4.5S RNA, encoded by the ffs gene, and at least one protein, Ffh, as well as a specific receptor on the membrane, called FtsY in E. coli, to which the SRP binds. FtsY is sometimes referred to as the docking protein because it “docks” proteins targeted by the SRP pathway to the SecYEG channel in the membrane. The ftsY (filament temperature-sensitive Y) gene was originally identified through temperature-sensitive mutations that cause E. coli not to divide properly and to form long filaments of many cells linked end to end at higher temperatures, but its role in cell division is indirect.

      Figure 2.38C illustrates how the SRP system works. The SRP binds to the first hydrophobic transmembrane sequence of an inner membrane protein as this region of the protein emerges from the ribosome. The complex binds to the membrane, and synthesis of the protein continues, feeding the protein directly into the SecYEG translocon as the protein emerges from the ribosome. The energy of translation due to cleavage of GTP to GDP drives the polypeptide out of the ribosome into the SecYEG translocon, replacing the role of SecA, although the SecA protein might still be required for transmembrane proteins with long periplasmic domains.

      The process of translating a protein as it is inserted into the translocon is called cotranslational translocation. There is a good reason why proteins destined for the inner membrane are cotranslated with their insertion into the translocon in the membrane while proteins targeted by the SecB pathway can first be translated in their entirety and then inserted into the translocon. Inner membrane proteins are much more hydrophobic than exported proteins and would form an insoluble aggregate in the aqueous cytoplasm if they were translated in their entirety before being transported into the membrane (see Lee and Bernstein, Suggested Reading).

      What happens after an inner membrane protein enters the SecYEG channel is less clear. The transmembrane domains of the protein must escape the SecYEG channel and enter the surrounding membrane, while the periplasmic and cytoplasmic domains must stay in the correct compartments. Presumably, the SecYEG channel has a lateral gate that opens and allows the transmembrane domains of the protein to escape into the membrane. Another inner membrane protein called YidC might help in this process (Figure 2.38C) (see Xie and Dalbey, Suggested Reading). YidC seems to be required for the lateral escape of some proteins but not others. Some inner membrane proteins bypass SecYEG altogether and require only YidC to enter the inner membrane.