Richard A. Rachubinski

A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase.

Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy‐termini. One such protein is the peroxisomal 3‐ketoacyl CoA thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600–4606]. Both rat peroxisomal thiolases are synthesized as larger precursors with an amino‐terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the thiolases into peroxisomes because expression of an altered cDNA encoding only the mature thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino‐terminal or internal locations and is distinct from the C‐terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix.

Cargo-Selected Transport from the Mitochondria to Peroxisomes Is Mediated by Vesicular Carriers

Mitochondria and peroxisomes share a number of common biochemical processes, including the beta oxidation of fatty acids and the scavenging of peroxides. Here, we identify a new outer-membrane mitochondria-anchored protein ligase (MAPL) containing a really interesting new gene (RING)-finger domain. Overexpression of MAPL leads to mitochondrial fragmentation, indicating a regulatory function controlling mitochondrial morphology. In addition, confocal- and electron-microscopy studies of MAPL-YFP led to the observation that MAPL is also incorporated within unique, DRP1-independent, 70-100 nm diameter mitochondria-derived vesicles (MDVs). Importantly, vesicles containing MAPL exclude another outer-membrane marker, TOM20, and vesicles containing TOM20 exclude MAPL, indicating that MDVs selectively incorporate their cargo. We further demonstrate that MAPL-containing vesicles fuse with a subset of peroxisomes, marking the first evidence for a direct relationship between these two functionally related organelles. In contrast, a distinct vesicle population labeled with TOM20 does not fuse with peroxisomes, indicating that the incorporation of specific cargo is a primary determinant of MDV fate. These data are the first to identify MAPL, describe and characterize MDVs, and define a new intracellular transport route between mitochondria and peroxisomes.

How proteins penetrate peroxisomes

Three decades after the unobtrusive debut of the peroxisome as a distinct subcellular organelle, biologists are paying attention to the special bag of tricks eukaryotic cells use to entice peroxisomal proteins from their site of synthesis in the cytosol to the peroxisome. In this minireview, we highlight some of the recent findings that have emerged, emphasize their significance, and contrast them with aspects of protein import into other subcellular destinations. Peroxisomal Targeting Signals The Beginning, the Middle, and the End Genetic and biochemical evidence has underscored the conservation of peroxisomal targeting signals (PTSs) from yeast to humans and led to the elucidation of at least two pathways for the transport of proteins to the peroxisome matrix (lumen). Each of these pathways is dependent on the molecular recognition of a specific PTS by its cognate receptor, which then hands off the protein to a putative translocation machinery housed in the peroxisome membrane. PTSl is a conserved C-terminal tripeptide (SKL or a variant) that constitutes the major targeting signal for proteins destined for the peroxisome matrix. In contrast, PTS2 is a conserved N-terminal nonapeptide (R/K)(L/V/I) (X)5(H/Q)(L/A) used by a smaller subset of peroxisomal matrix proteins. Other internally located PTSs have been described but remain poorly characterized (Purdue and Lazarow, 1994). Peroxisomal membrane proteins do not possess either PTSl or PTS2 sequences but are endowed instead with PTSs that have been defined only as fairly large internal segments of peroxisomal membrane proteins (Purdue and Lazarow, 1994). Despite the fact that all these PTSs are known to be necessary and sufficient for targeting to peroxisomes, a novel twist (addressed later) is the recent discovery that polypeptide chains devoid of a PTS can hitch a ride into peroxisomes by association with subunits that contain a PTS (Glover et al., 1994; McNew and Goodman, 1994). Omnipresent PTS Receptors Yeast and human cells selectively deficient in the PTS1 or PTS2 import pathway (or both) have been instrumental in the identification of PTS receptors. The earliest mutant discovered to be selectively compromised in the PTSl pathway alone was the pas8 mutant of Pichia pastoris. The protein, Pas8p, is tightly associated with the cytosolic face of the peroxisome membrane and is the PTSl receptor (PTSl R) (Terlecky et al., 1995; Figure 1). Minireview

Transcriptome profiling to identify genes involved in peroxisome assembly and function.

Yeast cells were induced to proliferate peroxisomes, and microarray transcriptional profiling was used to identify PEX genes encoding peroxins involved in peroxisome assembly and genes involved in peroxisome function. Clustering algorithms identified 224 genes with expression profiles similar to those of genes encoding peroxisomal proteins and genes involved in peroxisome biogenesis. Several previously uncharacterized genes were identified, two of which, YPL112c and YOR084w, encode proteins of the peroxisomal membrane and matrix, respectively. Ypl112p, renamed Pex25p, is a novel peroxin required for the regulation of peroxisome size and maintenance. These studies demonstrate the utility of comparative gene profiling as an alternative to functional assays to identify genes with roles in peroxisome biogenesis.

The life cycle of the peroxisome.

Peroxisomes are highly adaptable organelles that carry out oxidative reactions. Distinct cellular machineries act together to coordinate peroxisome formation, growth, division, inheritance, turnover, movement and function. Soluble and membrane-associated components of these machineries form complex networks of physical and functional interactions that provide supramolecular control of the precise dynamics of peroxisome biogenesis.

Mutants of the Yeast Yarrowia lipolytica Defective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis

ABSTRACT Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A andsrp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A,srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that inY. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54,PEX1, and PEX6 genes.

Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

Dynamics of peroxisome assembly and function.

Recent studies in human cells and in the yeast Yarrowia lipolytica have shown that peroxisomes consist of numerous structurally distinct subcompartments that differ in their import competency for various proteins and are related through a time-ordered conversion of one subcompartment to another. Our studies have implicated the fusion of small peroxisomal precursors as an early event in the multistep assembly of peroxisomes operating in Y. lipolytica. Newly discovered unexpected roles for peroxisomes in specific developmental programs have expanded the remarkable plasticity of peroxisomal functions. Here, we highlight recent discoveries on the highly dynamic nature of peroxisome assembly and function and suggest questions for future research in these areas.

Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.

The endoplasmic reticulum plays an essential role in peroxisome biogenesis

The field of peroxisome biogenesis has reached a crossroads. The results of recent investigations have necessitated a re-evaluation of current models of peroxisome assembly in which peroxisomes grow by the import of both membrane and matrix proteins from the cytosol and arise by fission of pre-existing peroxisomes. The ER has now been implicated as a central player in the peroxisome biogenetic pathway. Some peroxisomal membrane proteins pass through the ER en route to the peroxisome and exist as glycosylated species within the peroxisome. Vesicular elements containing peroxisomal proteins bud from the ER and, because of the action of specific proteins (probably Pex1p and Pex6p), fuse to form peroxisomes. Matrix proteins are imported into these vesicular elements but never pass through the ER. The movement of some vesicles from the ER to peroxisomes is regulated in an as yet unknown fashion by COPII proteins, while the retrograde pathway from the peroxisome back to the ER might involve recycling of ER-resident proteins by COPI vesicles. The establishment of systems that reconstitute peroxisome assembly in vitro will help to clarify the nature of the peroxisomal vesicular compartments, determine how they move away from and back to the ER, and identify the molecular players involved in their maturation into functional peroxisomes.