the E protein of phage λ into the phage head. The GroEL chaperonin consists of 14 identical polypeptides (7 making up each cylinder) of 60 kDa. Its cochaperonin cap is called GroES, which is also made up of 7 subunits, each 10 kDa in size. Unlike DnaK and the other chaperones, GroEL and GroES are required for E. coli growth, even at lower temperatures. The GroEL chaperonin is known to be required for folding of some essential proteins, which explains why GroEL is essential. The chaperonins come in two general types called the group I and group II chaperonins. The group I chaperonins, related to GroEL, are composed of 60-kDa subunits and are found in all bacteria and in the mitochondria and chloroplasts of eukaryotes; this makes sense, since these organelles are derived from bacteria (see the introductory chapter). These chaperonins and their cochaperonins are induced by heat shock and other stresses, so they are called the Hsp60 proteins and Hsp10 proteins for heat shock 60-kDa and 10-kDa proteins (see Bukau and Horwich, Suggested Reading). The group II chaperonins are found in the archaea and in the cytoplasm of eukaryotes. They have very little amino acid sequence in common with the group I chaperonins and are not composed of identical subunits (i.e., they are mixed multimers) and often have eight or more polypeptide subunits per cylinder. Furthermore, if they have a cochaperonin cap, it might be attached to the opening of the chamber rather than being detachable, like GroES. They may also be more dedicated and fold only a small subset of proteins, including actin in the eukaryotic cytoplasm. Nevertheless, the two types form similar cylindrical structures and presumably use a similar two-stroke mechanism to help fold proteins. Note that this is yet another example where the archaea and eukaryotes are similar to each other and different from the bacteria (see the introductory chapter).
Figure 2.37 Chaperonins. The GroEL (Hsp60)-type chaperonin multimers form two connected cylinders. A denatured or unfolded protein enters the chamber in one of the cylinders, and the chamber is capped by the cochaperonin GroES (Hsp10). The denatured protein can then be helped to fold in the chamber and is released. Entry of a second unfolded protein into the second cylinder helps to trigger release of the folded protein.
Protein Degradation
As noted above, protein folding can be coupled to protein degradation through the association of chaperones such as ClpA and ClpX with proteases such as ClpP. Complexes such as ClpAP and ClpXP are members of the family of ATP-dependent proteases, which use cleavage of ATP to provide the energy for protein unfolding and proteolysis (see Baker and Sauer, Suggested Reading). These enzymatic machineries are important not only for the destruction of misfolded and denatured proteins, but also for regulated proteolysis to target specific substrate proteins under specific conditions. Their activity can be directed by the presence of a specific degradation tag, a short protein sequence that increases affinity for a specific protease; these tags are similar to the sequence added by tmRNA to target incomplete proteins for degradation (see above). This tag may be present all the time within the target protein, but it becomes available for recognition only under certain conditions, which allows the target protein to be stable under some conditions and unstable under other conditions. Proteolysis can also be controlled by adaptor proteins that deliver specific target proteins to specific proteases. Regulated proteolysis is discussed as a mechanism for gene regulation in chapters 11 and 12.
The Actinobacteria, including important pathogens such as Mycobacterium tuberculosis, utilize a proteasome structure similar to that used in eukaryotes to mediate degradation of specific protein targets. This structure is barrel-shaped and carries out both ATP-dependent and ATP-independent proteolysis. Eukaryotes use a specific protein tag called ubiquitination to direct protein substrates to the proteasome; in Actinobacteria, this is replaced by a different protein modification called pupylation (see Becker and Darwin, Suggested Reading).
Protein Localization
About one-fifth of the proteins made in a bacterium do not remain in the cytoplasm and instead are transported or exported into or through the surrounding membranes. The terminology is often used loosely, but we refer to proteins that leave the cytoplasm as being transported. The process of transferring them through one or both membranes is secretion. If they are transferred through both membranes to the exterior of the cell, they are exported. Correspondingly, proteins that remain in either the inner or outer membrane are inner membrane proteins or outer membrane proteins, while those that remain in the periplasmic space are periplasmic proteins. Proteins that are passed all the way out of the cell into the surrounding environment are exported proteins.
By far the largest group of proteins that are transported from the cytoplasm are destined for the inner membrane. Inner membrane proteins often extend through the membrane a number of times and have some stretches that are in the periplasm and other stretches that are in the cytoplasm. The stretches that traverse the membrane have mostly uncharged, nonpolar (hydrophobic [see the inner cover]) amino acids, which make them more soluble in the membranes. A stretch of about 20 mostly hydrophobic amino acids is long enough to extend from one side of the bipolar lipid membrane to the other, and such stretches in proteins are called the transmembrane domains. The less hydrophobic stretches between them are called the cytoplasmic domains or periplasmic domains, depending on whether they extend into the cytoplasm on one side of the membrane or into the periplasm on the other side. Proteins with domains on both sides of the membrane are called transmembrane proteins and are very important, because they allow communication from outside the cell to the cytoplasm. Some transmemembrane proteins that play such a communicating role are discussed in chapter 12.
The Translocase System
Transported proteins usually contain many amino acids that are either polar or charged (basic or acidic), which makes it difficult for them to pass through the membranes. They must be helped in their translocation through the membrane by other specialized proteins. Some of these proteins form a channel in the membrane. Some transported proteins make their own dedicated channel, but most use the more general channel called the translocase, so named because its function is to translocate proteins.
A current picture of the structure of the translocase that helps proteins pass through the inner membrane, as well as how it works, is outlined in Figure 2.38. We can predict some of the features this channel must have. It must have a relatively hydrophilic inner channel through which charged and polar amino acids can pass. It also must normally be closed and should open only when a protein is passing through it; otherwise, other proteins and small molecules would leak in and out of the cell through the channel. Even the leakage of molecules as small as protons cannot be tolerated, because it would destroy the proton motive force. The channel is made up of one each of three proteins, SecY, SecE, and SecG, and is therefore called the SecYEG channel or SecYEG translocase. These three proteins form a heterotrimer made up of one each of the three different polypeptides. The SecY protein is by far the largest of the three proteins and forms the major part of the channel, while the other two proteins play more ancillary, albeit important, roles. One heterotrimer can form a large enough channel to let an unfolded protein through (see van den Berg et al., Suggested Reading), but it seems likely that more than one of these heterotrimers is involved.