Sorting of Peroxisomal Proteins

Peroxisomes are small organelles bounded by a single membrane. Unlike mitochondria and chloroplasts, peroxisomes lack DNA and ribosomes. Thus all peroxisomal proteins are encoded by nuclear genes, synthesized on ribosomes free in the cytosol, and then incorporated into preexisting or newly generated peroxisomes. As peroxisomes are enlarged by addition of protein (and lipid), they eventually divide, forming new ones, as is the case with mitochondria and chloroplasts.

The size and enzyme composition of peroxisomes vary considerably in different kinds of cells. However, all peroxi-somes contain enzymes that use molecular oxygen to oxidize various substrates, forming hydrogen peroxide (H2O2). Catalase, a peroxisome-localized enzyme, efficiently decomposes H2O2 into H2O. Peroxisomes are most abundant in liver cells, where they constitute about 1 to 2 percent of the cell volume.

Cytosolic Receptor Targets Proteins with an SKL Sequence at the C-Terminus into the Peroxisomal Matrix

The import of catalase and other proteins into rat liver per-oxisomes can be assayed in a cell-free system similar to that used for monitoring mitochondrial protein import (see Figure 16-25). By testing various mutant catalase proteins in this system, researchers discovered that the sequence Ser-Lys-Leu (SKL in one-letter code) or a related sequence at the C-terminus was necessary for peroxisomal targeting. Further, addition of the SKL sequence to the C-terminus of a normally cytosolic protein leads to uptake of the altered protein by peroxisomes in cultured cells. All but a few of the many different peroxisomal matrix proteins bear a sequence of this type, known as peroxisomal-targeting sequence 1, or simply PTS1.

The pathway for import of catalase and other PTS1-bearing proteins into the peroxisomal matrix is depicted in Figure 16-32. The PTS1 binds to a soluble receptor protein in the cytosol (Pex5), which in turn binds to a receptor in the peroxisome membrane (Pex14). The soluble and membrane-associated peroxisomal import receptors appear to have a function analogous to that of the SRP and SRP receptor in targeting proteins to the ER lumen. Still bound to Pex5, the imported protein then moves through a multimeric translocation channel, a feature that differs from protein import into the ER lumen. At some stage either during or after entry into the matrix, Pex5 dissociates from the peroxiso-mal matrix protein and is recycled back to the cytoplasm. In contrast to the N-terminal uptake-targeting sequences on proteins destined for the ER lumen, mitochondrial matrix, and chloroplast stroma, the PTS1 sequence is not cleaved from proteins after their entry into a peroxisome. Protein import into peroxisomes requires ATP hydrolysis, but it is not known how the energy released from ATP is used to power unidirectional translocation across the peroxisomal membrane.

The peroxisome import machinery, unlike most systems that mediate protein import into the ER, mitochondria, and chloroplast, can translocate folded proteins across the

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Peroxisomal matrix protein membrane. For example, catalase assumes a folded conformation and binds to heme in the cytoplasm before traversing the peroxisomal membrane. Cell-free studies have shown that the peroxisome import machinery can transport a wide variety of molecules, including very large ones. To explain this unusual ability, scientists have speculated that a translocation channel of variable size may assemble by an unknown mechanism to fit exactly the diameter of the PTS1-bearing substrate molecule and then disassemble once translocation has been completed.

A few peroxisomal matrix proteins such as thiolase are synthesized as precursors with an N-terminal uptake-targeting sequence known as PTS2. These proteins bind to a different cytosolic receptor protein, but otherwise import is thought to occur by the same mechanism as for PTS1-containing proteins.

Peroxisomal Membrane and Matrix Proteins Are Incorporated by Different Pathways

Autosomal recessive mutations that cause defective peroxisome assembly occur naturally in the human iU population. Such defects can lead to severe impairment of many organs and to death. In Zellweger syndrome and related disorders, for example, the transport of many or all proteins into the peroxisomal matrix is impaired; newly synthesized peroxisomal enzymes remain in the cytosol and

FIGURE 16-32 Import of peroxisomal matrix proteins directed by PTS1 targeting sequence. Step 1: Catalase and most other peroxisomal matrix proteins contain a C-termlnal PTS1 uptake-targeting sequence (red) that binds to the cytosolic receptor Pex5. Step |2|: Pex5 with the bound matrix protein interacts with the Pex14 receptor located on the peroxisome membrane. Step |3|: The matrix protein-Pex5 complex is then transferred to a set of membrane proteins (Pex10, Pex12, and Pex2) that are necessary for translocation into the peroxisomal matrix by an unknown mechanism. Step 4: At some point, either during translocation or in the lumen, Pex5 dissociates from the matrix protein and returns to the cytosol, a process that involves the Pex2/10/12 complex and additional membrane and cytosolic proteins not shown. Note that folded proteins can be imported into peroxisomes and that the targeting sequence is not removed in the matrix. [See P E. Purdue and P B. Lazarow, 2001, Ann. Rev. Cell Devel. Biol. 17:701; S. Subramani et al., 2000, Ann. Rev. Biochem. 69:399; and V. Dammai and S. Subramani, 2001, Cell 105:187.]

are eventually degraded. Genetic analyses of cultured cells from different Zellweger patients and of yeast cells carrying similar mutations have identified more than 20 genes that are required for peroxisome biogenesis. I

Studies with peroxisome-assembly mutants have shown that different pathways are used for importing peroxisomal matrix proteins versus inserting proteins into the peroxiso-mal membrane. For example, analysis of cells from some Zellweger patients led to identification of genes encoding the putative translocation channel proteins Pex10, Pex12, and Pex2. Mutant cells defective in any one of these proteins cannot incorporate matrix proteins such as catalase into perox-isomes; nonetheless, the cells contain empty peroxisomes that have a normal complement of peroxisomal membrane proteins (Figure 16-33b). Mutations in any one of three other genes were found to block insertion of peroxisomal membrane proteins as well as import of matrix proteins (Figure 16-33c). These findings demonstrate that one set of proteins translocates soluble proteins into the peroxisomal matrix but a different set is required for insertion of proteins into the peroxisomal membrane. This situation differs markedly from that of the ER, mitochondrion, and chloro-plast, for which, as we have seen, membrane proteins and soluble proteins share many of the same components for their insertion into these organelles.

Although most peroxisomes are generated by division of preexisting organelles, these organelles also can arise de novo by the two-stage process depicted in Figure 16-34. In this case, peroxisomal membrane proteins first are targeted to precursor membranes by sequences that differ from both PTS1 and PTS2. Analysis of mutant cells revealed that Pex19 is the receptor protein responsible for targeting of peroxisomal membrane proteins, while Pex3 and Pex16 are necessary for their proper insertion into the membrane. The insertion of peroxisomal membrane proteins generates membranes that have all the components necessary for import of matrix proteins, leading to the formation of mature,

(a) Wild-type cells

PMP70 Catalase

Stained for PMP70

Stained for catalase

(a) Wild-type cells

PMP70 Catalase

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Stained for catalase

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Peroxisome

(b) Pex1 mutants (deficient in matrix-protein import)

(b) Pex1 mutants (deficient in matrix-protein import)

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functional peroxisomes. Division of mature peroxisomes, which largely determines the number of peroxisomes within a cell, depends on still another protein, Pex11. Overexpression of the Pex11 protein causes a large increase in the number of peroxisomes, suggesting that this protein

M EXPERIMENTAL FIGURE 16-33 Fluorescent-antibody staining of peroxisomal biogenesis mutants reveals different pathways for incorporation of membrane and matrix proteins. Cells were stained with antibodies to PMP70, a peroxisomal membrane protein, or with antibodies to catalase, a peroxisomal matrix protein, then viewed in a fluorescent microscope. (a) In wild-type cells, both peroxisomal membrane and matrix proteins are visible as bright foci in numerous peroxisomal bodies. (b) In cells from a Pex12-deficient patient, catalase is distributed uniformly throughout the cytosol, whereas PMP70 is localized normally to peroxisomal bodies. (c) In cells from a Pex3-deficient patient, peroxisomal membranes cannot assemble, and as a consequence peroxisomal bodies do not form. Thus both catalase and PMP70 are mis-localized to the cytosol. [Courtesy of Stephen Gould, Johns Hopkins University.]

controls the extent of peroxisome division. The small per-oxisomes generated by division can be enlarged by incorporation of additional matrix and membrane proteins via the same pathways described previously.

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