Sec Systems of Archaea and Eukaryotes
Archaea and eukaryotes do not have SecB or SecA and use the SRP system to translocate all exported proteins. Although they lack SecA, they may have other systems that help direct already translated proteins to the translocon. The translocon itself was first discovered in eukaryotes and is composed of three proteins that form similar structures in all three kingdoms of life. The amino acid sequences of the SecY and SecE subunits are similar in all three kingdoms; only the sequence of the third subunit (SecG in bacteria) is very different in eukaryotes and archaea, where it may have different functions. While eukaryotes have other such channels, the translocase, which helps transported proteins to enter the endoplasmic reticulum of eukaryotic cells, is the one most similar to the SecYEG channel of bacteria.
The SRP system was also first described in eukaryotes, where it is much larger, consisting of a 300-nucleotide RNA and eight proteins, six in the SRP and two in the docking protein, called the SRP receptor. However, some of the proteins in eukaryotes are very similar to those in bacteria, such as the 54-kDa SRP protein in eukaryotes, which is similar to the Ffh protein in the SRP of bacteria. The SRP system of eukaryotes targets both membrane and presecretory proteins to the endoplasmic reticulum.
The Tat Secretion Pathway
Only proteins that have not yet folded and so are still long, flexible polypeptides can be transported by the narrow SecYEG channel. Once folded into their final three-dimensional structure, proteins are much too wide to fit through the channel. However, some proteins must fold in the cytoplasm before they can be transported. These include membrane proteins that contain redox factors, such as molybdopterin and FeS clusters, which are synthesized in the cytoplasm and can be inserted into the protein only after it has been folded. Other examples of proteins that are transported after they are folded are some heterodimers in which only one member has a signal sequence, so the other partner would be left behind if the signal sequencecontaining polypeptide is transported before the two polypeptides have combined and folded. Folded proteins and complexes are transported by the Tat system.
STRUCTURE OF THE TAT SYSTEM
The Tat system of E. coli has three subunits, TatA, TatB, and TatC, while that of B. subtilis has only two, with TatA and TatB seemingly combined into one larger subunit. In E. coli, TatB and TatC bind the signal peptide on the protein to be transported and then recruit TatA, which forms the channel in the membranes. In this way, the channel may form only when there is a protein to be transported. Unlike the Sec translocon, which uses both the energy of ATP cleavage by SecA and the proton motor force to drive the protein through the channel, the Tat system may use only the latter.
The Tat Signal Sequence
The signal sequence recognized by the Tat system is structurally similar to the signal sequence recognized by the Sec system, with a positively charged region followed by a longer hydrophobic region and a polar region, and is cleaved from the protein as it passes through the channel. However, it is somewhat longer, especially in the positively charged region. Two of the positively charged amino acids at the junction of the charged and hydrophobic regions are usually arginines, which give the Tat system its name (twin-arginine transport). The arginines are found in the motif S-R-R, although the first of the twin arginines is sometimes a lysine (K). This sequence is followed by two hydrophobic amino acids, usually F and L, and then often by a K.
The presence of this particular signal sequence at the N terminus of a newly synthesized protein targets the protein for transport by the Tat system rather than by the SecYEG channel. This raises an interesting question. How does the system know the protein has already folded properly and contains all the needed cofactors, etc., so that it is time to transport it? The Tat system needs a “quality control” system to ensure that it transports only properly folded proteins and does not transport proteins that are unfolded or only partially folded. This quality control system should also be specific for each protein to be transported, since each type of folded protein has a unique structure (see above). E. coli solves this problem by encoding dedicated proteins that specifically bind to the Tat signal sequence of only one type of protein and come off only when that protein has folded properly (see Palmer et al., Suggested Reading).
Tat Systems in Other Organisms
Most bacteria and archaea, as well as the chloroplasts of plants (which descended from cyanobacteria [see the introduction]), have a Tat secretion system, although they might differ from that of E. coli in the number of subunits. Some bacteria, including B. subtilis, have two or more Tat systems. Some of them are dedicated to the transport of only one or very few proteins.
Disulfide Bonds
Another characteristic of proteins that are exported to the periplasm or secreted outside the cell is that many of them have disulfide bonds between cysteines (see the inside front cover). In other words, two of the cysteines in the protein are held together by covalent bonds between their sulfides. The sulfur atom of a cysteine in a disulfide bond is in its oxidized form because one of its electrons is shared by the two sulfurs, while the sulfur atom of an unbound cysteine is in its reduced form because it has an extra electron. These disulfide bonds can be between two cysteines in the same polypeptide chain or between cysteines in different polypeptide chains. Exported proteins need the covalent disulfide bonds to hold them together in the harsh environments of the periplasm and outside the cell. Failure to form the correct disulfide bonds or formation of disulfide bonds between the wrong cysteines can result in inactivity of the protein.
The disulfide bonds in proteins are formed by enzymes called disulfide oxidoreductases (DsbA, DsbB, etc.) as the proteins pass through the oxidizing environment in the periplasmic space between the inner and outer membranes of bacteria that contain outer membranes (or at the outer cell surface of bacteria that lack the outer membrane and periplasm). Proteins that are found inside the cell in the cytoplasm lack disulfide bonds because of the “reducing atmosphere” inside the cytoplasm due to the presence of high concentrations of small reducing molecules, such as glutathione, thioredoxin, and bacillithiol. In fact, the appearance of disulfide bonds in some cytoplasmic regulatory proteins is taken as a signal by the cell that oxidizing chemicals are accumulating in the cell and that proteins should be made to combat the potentially lethal oxidative chemical stress.
Protein Secretion and Export
Some proteins are transported through the membrane(s) to the outside of the cell, where they can remain attached, enter the surrounding medium, or even directly enter another cell. The process differs markedly between cells that have or lack an outer membrane. Bacteria that have an outer membrane require translocation from the periplasm through the extremely hydrophobic outer membrane. Because of the additional challenge created by the outer membrane, these bacteria have developed elaborate specialized structures to export proteins. Many of these play important roles in bacterial pathogenesis, so they have attracted considerable attention.
Protein Secretion Systems in Bacteria with an Outer Membrane
At least six basic types of protein secretion systems, imaginatively named types I to VI, have been identified. All of these secretion systems rely on channels in the outer membrane (called β-barrels or secretins) formed from β-sheets organized in a ring (see Figure 2.20 for an explanation of protein secondary and tertiary structures). The β-barrels are assembled so that the side chains of charged and polar amino acids tend to be in the center of the barrel, where they are in contact with hydrophilic proteins that are passing through, while the side chains of hydrophobic amino acids are on the outside of the barrel in contact with the very hydrophobic surrounding membrane. Assembly of the β-barrels requires a complex of proteins called the Bam complex (BamA, -B, -C, etc.) and periplasmic chaperones, including Skp.