Posttranslational Targeting of Proteins

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CHAPTER 18 Posttranslational Targeting of Proteins*

Protein synthesis is largely a monopoly of cytoplasmic ribosomes that provide all of the proteins for the nucleus, cytoplasm, peroxisomes, and secretory pathway. Even mitochondria and chloroplasts import most of their proteins from cytoplasm, despite the fact that they originated as bacterial endosymbionts and have retained the capacity to synthesize a few of their proteins. Most of the original bacterial genes moved to the nucleus of the eukaryotic host.

Given a common site of synthesis, accurate addressing is essential to direct proteins to their sites of action and to maintain the unique character of each cellular compartment. This is achieved by “zip codes” built into the structure of each protein (Fig. 18-1). Residues in the sequence of each protein—often, but not necessarily, contiguous amino acids—form a signal for targeting.

Targeting signals are both necessary and sufficient to guide proteins to their final destinations. Transplantation of a targeting signal, such as a presequence from a mitochondrial protein, to a cytoplasmic protein reroutes the hybrid protein into the organelle specified by the targeting sequence, mitochondria in this example. Some targeting signals are transient parts of the protein. For example, most mitochondrial proteins are synthesized with N-terminal extensions that guide them to mitochondria and then are removed. Alternatively, signals may be a permanent part of the mature protein, in some cases serving repeatedly to target a mobile protein between different destinations. Permanent nuclear targeting signals can be located at the N-terminus, the C-terminus, or even the middle of a protein. Some proteins have more than one targeting signal: a primary code that directs the protein to the target organelle or pathway, and a second signal that steers the protein to its specific site of residence within the organelle or pathway.

Targeting signals direct proteins to their destination by binding to organelle-specific receptors or using soluble “escort” factors as intermediaries. When necessary, proteins cross membranes via channels called translocons formed by integral membrane proteins (Fig. 18-2). Like ion channels (see Chapter 10), these protein-translocating channels are gated to prevent indiscriminate transport of cellular constituents when not occupied by a polypeptide. Polypeptides fit so tightly in these channels during translocation that ions do not leak through. Ions traverse ion channels in a microsecond, whereas polypeptides take tens of seconds to move through translocons. Protein synthesis, adenosine triphosphate (ATP) hydrolysis, or the membrane potential provides the energy to power protein translocation across membranes.

Three families of protein translocation channels are found in all three domains of life. Sec translocons direct proteins into the endoplasmic reticulum in eukaryotes and out of prokaryotes. The Tat family of pores translocate folded proteins into chloroplast thylakoids and out of prokaryotes. Membrane proteins related to Oxa1p help to insert proteins synthesized in the mitochondrial matrix and prokaryotic cytoplasm into membranes. Mitochondria (Fig. 18-4), chloroplasts (Fig. 18-6), and prokaryotes (Fig. 18-10) have additional families of protein translocation channels.

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Figure 18-10 secretion across the outer membrane of gram-negative bacteria. A, Pathways dependent on SecYE. The cleaved signal sequence is shown in blue. The β-domain of autotransporters forms a pore for the translocation of part of its own chain, which may remain attached, as shown, or be cleaved for escape from the cell. Single accessory proteins form a pore for secretion of separate proteins. Usher forms a pore for the translocation and assembly of pili. Type II secretion uses a secretin pore for translocation. Type IV secretion employs a large translocon similar to that used by Agrobacterium for secretion of DNA. B, Pathways independent of SecYE. Type I secretion uses an ABC transporter to cross the inner membrane and additional subunits to cross the periplasm and outer membrane. Left panel, Ribbon model of TolC, one type of translocon that spans the periplasm and outer membrane. Right panel, Each TolC subunit contributes four β-strands to a porin-like structure that spans the outer membrane. α-Helical continuations of these β-strands form a tube having an internal diameter of 3.5 nm for transport of proteins across the periplasm. Bacterial flagella transport flagellin subunits across both membranes and then through the central channel of the flagellar filament for incorporation at the growing tip. Type III secretion uses components similar to the basal body of flagella. Gray illustration (far right) shows a three-dimensional reconstruction of the type III secretion apparatus from Salmonella typhimurium. IM, inner membrane; OM, outer membrane.

(A–B, Drawings based on Thanassi DG, Hultgren SJ: Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 12:420–430, 2000. B, TolC ribbon diagram based on PDB file: 1EK9. Reference: Koronakis V, Sharff A, Koronakis E, et al: Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405:914–919, 2000. Reconstruction of the type III secretion complex from S. typhimurium based on Marlovits TC, Kubori T, Sukhan A, et al: Structural insights into the assembly of the type II secretion needle complex. Science 306:1040–1042, 2004.)

Primary targeting can occur either cotranslationally, coincident with protein synthesis, or posttranslationally, after polypeptide synthesis. Chapter 20 covers protein targeting to endoplasmic reticulum where, with a few exceptions, targeting is cotranslational. This chapter covers posttranslational targeting mechanisms that move proteins across membrane bilayers into mitochondria, chloroplasts, and peroxisomes and out of Bacteria. Eukaryotes also secrete a few proteins directly across the plasma membrane. Chapter 14 covers posttranslational movements of proteins into and out of the nucleus through a large aqueous channel in the nuclear pore.

Transport of Proteins into Mitochondria

Mitochondrial outer and inner membranes define two spaces: one between the outer and inner membranes (intermembranous space) and an interior space termed the matrix (Fig. 18-3). Each membrane and space has distinct functions and protein compositions, which are covered in Chapter 19. Targeting signals and specific translocation machinery guide more than 500 imported proteins selectively to these compartments.

Genetic and biochemical experiments on fungi defined the molecular machinery for proteins to enter mitochondria, including the Tom complex (translocase of the outer mitochondrial membrane), the Sam complex (sorting and assembly machinery of the outer membrane), and two Tim complexes (translocase of the inner mitochondrial membrane). See Figures 18-4 and 18-5. Although the distinction is not absolute, one Tim complex is specialized to transport proteins into the matrix, and the other is specialized for insertion of proteins into the inner membrane. Translocation requires energy and assistance from protein chaperones both outside and inside mitochondria.

Delivery of Protein to Mitochondria

After synthesis by cytoplasmic ribosomes, most proteins destined for mitochondria bind cytosolic chaperones of the Hsp70 family (see Fig. 17-14). This interaction maintains proteins in unfolded configurations competent for import. Some imported proteins require additional factors, such as mitochondria-import stimulation factor, for targeting to the translocation machinery.

Targeting signals for proteins of the matrix are generally located at the N-termini of precursor polypeptides as contiguous sequences of 10 to 70 amino acids. These targeting motifs are called presequences, because they are usually removed by proteolytic cleavage in the mitochondrial matrix. Presequences are rich in basic, hydroxylated, and hydrophobic amino acids but share no sequences in common. The targeting sequences of many mitochondrial membrane proteins are in the middle of the polypeptide and are not cleaved after import. Cytochrome c, a component of the electron transport chain in the intermembranous space (see Fig. 19-5), also has an internal signal for import into mitochondria.

A succession of weak interactions with outer membrane receptors Tom20, Tom22, Tom5, and perhaps Tom70 guide presequences and other target signals to the outer membrane translocon. The presequence initially contacts Tom20. Eight residues of the presequence fold into an amphipathic (hydrophobic on one side, hydrophilic on the other) α-helix that binds in a shallow hydrophobic groove on Tom20. Arginines on the surface of this helix interact with acidic residues on Tom22 (Fig. 18-4D). Other parts of the presequence are thought to interact with Tom40, the translocon itself. Although these associations are weak, collectively, they distinguish mitochondrial presequences from other proteins in the cytoplasm with high fidelity.

Translocation across the Outer Membrane

Outer membrane receptors transfer the presequence to the translocon channel, which is composed mainly of Tom40 along with three small subunits. Tom40 is an integral membrane protein that is predicted to span the bilayer exclusively as β-strands. Electron microscopy of purified Tom complex revealed two pores with diameters of approximately 2 nm, which agrees with the size of the pore calculated from ion conductance measurements of purified Tom40 inserted into lipid bilayers. Two molecules of Tom40 are postulated to form a channel and the complex may contain two or three of these channels. Proteins must be largely unfolded to fit through a pore of this size. Like Sec translocons of endoplasmic reticulum (see Fig 20-6) and bacteria (Fig. 18-9), Tom channels are likely to be gated, so they close when not occupied by a translocating polypeptide. After crossing the outer membrane, some proteins remain in the intermembranous space.

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Figure 18-9 Secretion of proteins from bacteria through the SecYE translocon. A, Pathway of secretion. 1, After synthesis by a cytoplasmic ribosome, the polypeptide associates with the SecB chaperone. 2, SecA binds the presequence (blue) and docks on the SecYE translocon. 3, The presequence inserts into the translocon. 4, ATP-binding to SecA promotes insertion of the associated polypeptide into the translocon, followed by cleavage of the signal sequence. 5–7, The membrane potential and cycles of ATP hydrolysis by SecA drive the polypeptide across the inner membrane. B, Ribbon diagram of Haemophilus influenzae SecB. C, Ribbon diagram of Bacillus subtilis SecA. D, Ribbon diagram of Methanococcus jannaschii SecY complex translocon.

(A, Modified from Danese PN, Silhavy TJ: Targeting and assembly of periplasmic and outer-membrane proteins in E. coli. Annu Rev Genet 32:59–94, 1999. B, PDB file: IOZB. Reference: Zhou J, Xu Z: Structural determinants of SecB recognition by SecA in bacterial protein translocation. Nature Struct Biol 10:942–948, 2003. C, PDB file: 1TF2. Reference: Osborne AR, Clemons WM, Rapoport TA: A large conformational change of the translocation ATPase SecA. PNAS 101:10937–10942, 2004. D, PDB file: 1RHZ. Reference: van de Berg B, Clemons WM, Collinson I, et al: X-ray structure of a protein-conducting channel. Nature 427:36–44, 2004.)