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 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.
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.
Assembly of Outer Membrane Proteins
Some simple outer membrane proteins transfer laterally into the bilayer while they are in transit through Tom, while more complicated outer membrane proteins, including Tom40 itself and porins (see Fig. 7-8), require assistance. Two protein complexes of the outer membrane called Sam I and Sam II mediate folding and insertion into the membrane.
Translocation across the Inner Membrane to the Matrix
Proteins use the Tim23 translocon to cross the inner membrane into the matrix. The channel across the inner membrane is formed by the integral membrane proteins Tim23 and Tim17 (Fig. 18-4
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