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Peroxisome assembly in yeast.

01 Jun 2003-Microscopy Research and Technique (Wiley)-Vol. 61, Iss: 2, pp 139-150
TL;DR: This review summarizes relevant recent data and the progress in the understanding of the principles of the peroxisomal matrix protein import machinery is discussed.
Abstract: Peroxisomes are essential organelles that may be involved in various functions, dependent on organism, cell type, developmental stage of the cell, and the environment Until recently, peroxisomes were viewed as a class of static organelles that developed by growth and fission from pre-existing organelles Recent observations have challenged this view by providing evidence that peroxisomes may be part of the endomembrane system and constitute a highly dynamic population of organelles that arises and is removed upon environmental demands Addi- tionally, evidence is now accumulating that peroxisomes may arise by alternative methods This review summarizes relevant recent data on this subject In addition, the progress in the under- standing of the principles of the peroxisomal matrix protein import machinery is discussed Microsc Res Tech 61:139 -150, 2003 © 2003 Wiley-Liss, Inc

Summary (4 min read)

INTRODUCTION

  • Microbodies (peroxisomes, glyoxysomes, and glycosomes) are morphologically simple organelles.
  • The functional diversity of these organelles is unprecedented and varies with the organism in which they occur.
  • Thus, microbodies may be involved in such distinct functions as carbon catabolism in fungi, biosynthetic processes (e.g., synthesis of amino acids and penicillin in fungi, and of cholesterol in man), photorespiration in plants, or glycolysis in Trypanosomes (Borst, 1989; Van den Bosch et al., 1992; Veenhuis and Harder, 1988).
  • Their understanding of the molecular principles of assembly and function of the organelle is still limited.
  • Here, the authors will highlight recent observations on fungal peroxisomes and discuss the current working models on the biogenesis of these intriguing organelles.

PEROXISOME PROLIFERATION

  • A characteristic feature of yeast peroxisomes is that they are inducible.
  • When grown at high dilution rates, H. polymorpha cells generally contained a single large peroxisome that harbored the enzymes alcohol oxidase (AO), dihydroxyacetone synthase (DHAS), and catalase (CAT).
  • Taken together, these data leave little doubt that it is not the matrix protein levels but rather the environmental conditions that prescribe the ultimate number and size of peroxisomes, and, thus, control the multiplication of the organelle.
  • The Netherlands Organization for the Advancement of Pure Research, Earth and Life Sciences (NWO/ALW), also known as Grant sponsor.
  • The authors suggested that the oligomeric state of Pex11p could play a role in peroxisome maturation as the protein was found as monomer in small, newly formed organelles and as dimer in mature ones.

PEROXISOME BIOGENESIS

  • Genes Involved in Peroxisome Biogenesis Molecular approaches to identify proteins essential for peroxisome biogenesis became feasible in the early 1990s when viable yeast mutants were isolated that were defective in peroxisome development and function (collectively called pex mutants; see Distel et al. (1996) for the unified nomenclature).
  • Yeast pex mutants have lost the capacity to grow on carbon sources (e.g., fatty acids, methanol)—but not nitrogen sources (e.g., primary amines)—that are metabolized by peroxisomal enzymes.
  • Corresponding genes were cloned by functional complementation of these mutants, using genomic or cDNA libraries.
  • At present, 23 different PEX genes from various yeasts are now identified (H. polymorpha, S. cerevisiae, P. pastoris, and Yarrowia lipolytica; (reviewed by Subramani, 1998; for a recent listing see http://www.peroxisome. org/).
  • Most PEX gene products (termed peroxins) that have been identified so far are thought to play a role in matrix protein import and are discussed below.

Matrix Protein Import Signals

  • Peroxisomal matrix proteins are encoded by nuclear genes and are synthesized in the cytosol on free ribosomes (Lazarow and Fujiki, 1985).
  • So far, two peroxisomal targeting signals have been characterized that mediate sorting of the protein to peroxisomes (De Hoop and AB, 1992; Rachubinski and Subramani, 1995; Subramani, 1998).
  • The PTS1 consensus sequence is –SKL.COOH, but various variants of this motif are allowed (Gould et al., 1989; Lametschwandtner et al., 1998).
  • Most likely other, possibly internal, signals also exist for a subset of specific proteins, e.g., malate synthase and acyl CoA synthase (Bruinenberg et al.

PTS1 Protein Import: 1. Receptor Binding to Matrix Proteins

  • The initial data on the location of the PTS1-receptor, Pex5p, in different organisms were conflicting and varied with the organisms and/or the experimental approaches used.
  • This has led to different models for the mechanisms of PTS1 protein import.
  • In H. polymorpha, Pex5p is localized in the cytosol and in the peroxisomal matrix; the putative membrane-bound portion of the protein is invariably below the limit of detection (Van der Klei et al., 1995, 1998).
  • This would implicate that a protein export mechanism must exist for peroxisomes.
  • Furthermore, Lametschwandtner et al. (1998) showed that additional targeting information could be present in the residues preceding the PTS1 in matrix proteins, which most probably modulate the strength of the interaction of the cargo protein with Pex5p.

PTS1 Protein Import: 2. Receptor-Cargo Docking to the Peroxisomal Membrane

  • Upon binding a PTS1, the receptor-cargo complex interacts at a putative docking site on the peroxisomal membrane en route to the peroxisomal matrix.
  • Apparently, additional components (e.g., peroxins, PTS1 cargo proteins), other domains of Pex14p or the recently reported phosphorylation of Pex14p (Johnson et al., 2001; Komori et al., 1999) may influence the Pex5p-Pex14p binding event.
  • Schliebs et al. (1999) furthermore showed that one Pex5p molecule contains 5–7 binding sites for the N-terminal Pex14p fragment.
  • On the basis of this differential import phenomenon, Kiel and Veenhuis (2000) concluded that apparently some PTS1 proteins are completely dependent on Pex14p whist others do not require the function of Pex14p to get imported under conditions that sufficient Pex5p is available.
  • On the basis of their data, Urquhart et al. (2000) suggested an alternative role for Pex13p in a later stage of the process that occurs after docking.

PTS1 Protein Import: 3. Import Into the Organelle/Release of the Cargo

  • How matrix proteins actually enter peroxisomes or pre-peroxisomal structures, as described for Y. lipolytica (see below), is yet totally unclear.
  • Several authors have proposed that the zincbinding (RING finger) proteins, Pex10p and Pex12p, eventually together with the third zinc-finger protein Pex2p, may function in the translocation process (Chang et al., 1999, reviewed in Holroyd and Erdmann, 2001).
  • Furthermore, recently Albertini and coworkers (2001) demonstrated that a RING finger complex is associated with the docking complex.
  • These data suggest that Pex8p functions at an intra-organellar stage of the PTS1 protein import cascade.

PTS1 Protein Import: 4. Recycling of Pex5p to the Cytosol

  • A peroxin that is proposed to be involved in a late stage of Pex5p-dependent protein import is the ubiquitin-conjugating enzyme Pex4p (Crane et al., 1994; Van der Klei et al., 1998; Wiebel and Kunau, 1992).
  • Remarkably, in pex4 cells part of the overproduced Pex5p accumulated at the inner surface of the peroxisomal membrane (Van der Klei et al., 1998).
  • Apparently, CAT and MAS require additional factors to become imported and that were not overproduced.
  • It must be noted that these proteins have been implicated in vesicle fusion processes as well (see below).
  • Furthermore, overexpression of PEX5 in H. polymorpha pex1 and pex6 mutants does not rescue the PTS1 import deficiency (Salomons et al., 2000).

Import of PTS2 Containing Matrix Proteins

  • In fungi, matrix proteins containing a PTS2 sequence are very rare.
  • It must be noted that for Pex7p no interactions with Pex12p or Pex8p have been reported, although these genes are required for PTS2 import.
  • Also in baker’s yeast, the accessory proteins Pex18p and Pex21p are required to import thiolase by binding directly to Pex7p (Purdue et al., 1998).
  • Thus, so far the data concerning PTS2 import are relatively scarce.

PEROXISOMAL MATRIX PROTEIN ASSEMBLY

  • Little is known of the mechanisms involved in the assembly of peroxisomal matrix proteins.
  • There is now convincing evidence that matrix proteins may be transported into the organelle in their mature form.
  • The observation that AO is imported as monomers is not unexpected for physiological reasons.
  • Possibly, Pex20p acts as a cytosolic chaperone assisting oligomerisation of thiolase into dimers that normally may occur prior to its import into the peroxisome (Titorenko et al., 1998).
  • C. boidinii pmp47 mutants mislocalize inactive DHAS molecules to the cytosol.

CAN PEROXISOMES DEVELOP FROM THE ENDOMEMBRANE SYSTEM?

  • Upon their discovery, peroxisomes were thought to develop by budding from the ER (De Duve and Baudhuin, 1966).
  • In yeast, there is solid evidence that this model is true under conditions of normal peroxisome induction.
  • In addition, in other organisms comparable mechanisms have not been described yet.
  • The authors subsequently noticed that synthesis of a fusion protein containing the initial 50 N-terminal amino acids of Pex3p fused to GFP led to the formation of numerous small vesicles that were located at a distinct site of the nuclear membrane.
  • Taken together, these data indicate that peroxisome formation may follow different pathways, namely the “classical” way by growth and fission and alternative ways, using the endomembrane system as template.

CONCLUDING REMARKS

  • In the last decade, many of the protein components involved in peroxisome biogenesis (PEX genes) have been identified and much learned about their putative function, localization, and possible interaction with other proteins.
  • The principles of the matrix protein import machinery are unsolved.
  • On the other hand, essential components may still be missing.
  • To understand the function of the peroxisomal membrane, analysis of its transport properties and the proteins involved is needed.
  • As mutants affected in these genes most likely do not have a pex phenotype, adapted mutant screens have to be designed or alternatively, transport proteins should be identified by alternative ways (screening of databases, purification of proteins) and their genes cloned by reverse genetics.

ACKNOWLEDGMENTS

  • Ida van der Klei holds a PIONIER grant from the Netherlands Organization for the advancement of Pure Research (NWO/ALW).
  • Jan Kiel is supported by a grant from ALW, which is subsidized by the Dutch Organization for the Advancement of pure Research (NWO).
  • The authors thank Anna Rita Bellu, Ralf van Dijk, and Klaas Nico Faber for providing data prior to publication.

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University of Groningen
Peroxisome assembly in yeast
Veenhuis, M; Kiel, JAKW; Van der Klei, IJ
Published in:
Microscopy Research and Technique
DOI:
10.1002/jemt.10323
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Microscopy
Research and Technique
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Peroxisome Assembly in Yeast
MARTEN VEENHUIS,
*
JAN A.K.W. KIEL, AND IDA J. VAN DER KLEI
Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, NL-9750
AA Haren, The Netherlands
KEY WORDS yeast; peroxisome biogenesis; PEX genes; protein translocation
ABSTRACT Peroxisomes are essential organelles that may be involved in various functions,
dependent on organism, cell type, developmental stage of the cell, and the environment. Until
recently, peroxisomes were viewed as a class of static organelles that developed by growth and
fission from pre-existing organelles. Recent observations have challenged this view by providing
evidence that peroxisomes may be part of the endomembrane system and constitute a highly
dynamic population of organelles that arises and is removed upon environmental demands. Addi-
tionally, evidence is now accumulating that peroxisomes may arise by alternative methods. This
review summarizes relevant recent data on this subject. In addition, the progress in the under-
standing of the principles of the peroxisomal matrix protein import machinery is discussed.
Microsc. Res. Tech. 61:139 –150, 2003.
© 2003 Wiley-Liss, Inc.
INTRODUCTION
Microbodies (peroxisomes, glyoxysomes, and glyco-
somes) are morphologically simple organelles. They
consist of a single membrane that encloses a protein-
aceous matrix and measure up to 1 m in diameter.
Despite their simple morphology, the functional diver-
sity of these organelles is unprecedented and varies
with the organism in which they occur. Thus, micro-
bodies may be involved in such distinct functions as
carbon catabolism in fungi, biosynthetic processes (e.g.,
synthesis of amino acids and penicillin in fungi, and of
cholesterol in man), photorespiration in plants, or gly-
colysis in Trypanosomes (Borst, 1989; Van den Bosch et
al., 1992; Veenhuis and Harder, 1988). Although many
of the players that are involved in peroxisome biogen-
esis have now been identified, our understanding of the
molecular principles of assembly and function of the
organelle is still limited. So far, the data lend support
to the view that the strategies that the cell uses to
mediate peroxisome development may, at least in part,
differ between various organisms. Here, we will high-
light recent observations on fungal peroxisomes and
discuss the current working models on the biogenesis of
these intriguing organelles.
PEROXISOME PROLIFERATION
A characteristic feature of yeast peroxisomes is that
they are inducible. The organelles rapidly develop dur-
ing adaptation of cells to a new environment that re-
quires one or more peroxisomal enzymes for growth.
Growth substrates known to induce peroxisomes in
various yeasts include alkanes, oleic acid, methanol,
D-amino acids, purines, and primary amines (reviewed
by Veenhuis and Harder, 1988). Remarkably, the num-
ber and volume fraction of peroxisomes is not pre-
scribed by the amount of protein that is accommodated
but, instead, appears to be predominantly determined
by the growth conditions, i.e., the choice of the sub-
strate, the growth rate, and the oxygen concentration
(Veenhuis and Harder, 1988). Overproduction of spe-
cific matrix proteins in WT cells made it clear that the
maximal protein storage capacity of peroxisomes is
normally not used (Distel et al., 1988; Godecke et al.,
1989). Even in cells that contain identical sets of per-
oxisomal matrix proteins, the size and number of the
organelles may strongly differ. This was elegantly dem-
onstrated in glucose-limited continuous cultures of
Hansenula polymorpha. When grown at high dilution
rates, H. polymorpha cells generally contained a single
large peroxisome that harbored the enzymes alcohol
oxidase (AO), dihydroxyacetone synthase (DHAS), and
catalase (CAT). At low dilution rates, however, several
smaller organelles were present of identical protein
composition (Fig. 1). Also, in H. polymorpha Pim mu-
tants, in which a major portion of the peroxisomal
matrix proteins resides in the cytosol, small peroxi-
somes were present at numbers equal or even exceed-
ing those of WT cells (Waterham et al., 1992b). Taken
together, these data leave little doubt that it is not the
matrix protein levels but rather the environmental
conditions that prescribe the ultimate number and size
of peroxisomes, and, thus, control the multiplication of
the organelle. So far, the highest peroxisome induction
rates have been encountered in methylotrophic yeast
species (e.g., Candida boidinii, Hansenula polymor-
pha, Pichia pastoris). When H. polymorpha is grown in
methanol-limited chemostat cultures at low dilution
rates, up to 80% of the total cytoplasmic volume of the
cells may be occupied by peroxisomes (Fig. 2). Remark-
ably, these organelles display a crystalline substruc-
ture due to the crystallization of AO protein (Veenhuis
et al., 1978).
Grant sponsor: The Netherlands Organization for the Advancement of Pure
Research, Earth and Life Sciences (NWO/ALW).
*Correspondence to: Prof. Dr. M. Veenhuis, Eukaryotic Microbiology, Gro-
ningen Biomolecular Sciences and Biotechnology Institute (GBB), University of
Groningen, P.O. Box 14, NL-9750 AA Haren, The Netherlands.
E-mail: M.Veenhuis@biol.rug.nl
Received 12 August 2001; accepted in revised form 18 February 2002
DOI 10.1002/jemt.10323
Published online in Wiley InterScience (www.interscience.wiley.com).
MICROSCOPY RESEARCH AND TECHNIQUE 61:139 –150 (2003)
© 2003 WILEY-LISS, INC.

Kinetics experiments, using H. polymorpha cells, re-
vealed an unexpected characteristic of their peroxi-
somes, namely that these organelles are only tempo-
rally matrix protein import-competent. Small peroxi-
somes grow because of the incorporation of proteins
and lipids. At a certain size, determined by the growth
conditions (see below), growth ceases and one, or infre-
quently few, new organelles are formed by ssion from
the mature one, which in turn start to grow during
prolonged cultivation (Fig. 3A,B). Apparently, these
small organelles have inherited the capacity to grow
from the mature parent, leaving this organelle as an
enzyme bag that is no longer capable of protein up-
take but remains physiologically active (Veenhuis et
al., 1989; Waterham et al., 1992a). The mechanisms
that control peroxisome maturation and proliferation
are largely unknown. However, studies on the function
of the peroxisomal membrane protein Pex11p indicate
that this protein might play a role in organelle multi-
plication. Overexpression of Pex11p results in prolifer-
ation of peroxisomes while its absence results in the
formation of giant peroxisomes, causing problems to
distribute peroxisomes over mother and daughter cells
during cell division (Erdmann and Blobel, 1995; Mar-
shall et al., 1995). Indeed, the group of Goodman
showed that Pex11p induction in vivo resulted in frag-
mentation of large peroxisomes into smaller organelles
(Marshall et al., 1996). The authors suggested that the
oligomeric state of Pex11p could play a role in peroxi-
some maturation as the protein was found as monomer
in small, newly formed organelles and as dimer in
mature ones. Recently, however, a direct role for
Pex11p in -oxidation in Saccharomyces cerevisiae was
proposed. Van Roermund and coworkers (2000) con-
cluded that Pex11p is required to transport medium
chain fatty acids into peroxisomes. In their view, its
effect on peroxisome proliferation is only indirect re-
sulting from an increased -oxidation. Although such a
scenario may explain the results obtained in S. cerevi-
siae, the proliferation effect upon Pex11p overproduc-
tion observed in, e.g., Trypanosomes that mainly play a
role in glycolysis (Lorenz et al., 1998), cannot be recon-
ciled with an exclusive role for Pex11p in fatty acid
transport. Clearly, more research is required to un-
ravel the mechanisms controlling peroxisome matura-
tion/proliferation.
Fig. 1. Comparison of the size and number of peroxisomes in cells, grown in glucose-limited chemo-
stats at different dilution rates. A: D 0.18 h
-1
. B: D 0. 05 h
-1
. (For all gures, electron micrographs
are taken of WT H. polymorpha cells, xed with KMnO
4
unless otherwise stated. Abbreviations: N,
nucleus; P, peroxisome; V, vacuole. Scale bars 0.5 m.)
Fig. 2. Typical example of a cell grown in a methanol-limited
chemostat (D 0.1 h
-1
), crowded with peroxisomes.
140 M. VEENHUIS ET AL.

Peroxisomes that have become redundant for
growth, i.e., upon a shift of cells from peroxisome in-
ducing (e.g., methanol) to peroxisome-repressing (e.g.,
glucose) conditions, are degraded by a selective process
designated pexophagy (reviewed by Bellu and Kiel,
2003; see this issue). Studies in H. polymorpha have
suggested that in particular mature organelles be de-
graded leaving the small, import-competent ones unaf-
fected. Recently, we showed that a protein involved in
the biogenesis of peroxisomes, the peroxin Pex14p, is
also involved in pexophagy and may act as a molecular
switch that discriminates between the import compe-
tence and incompetence of individual organelles (Bellu
et al., 2001). The physiological advantage of this mech-
anism is immediately clear because it allows the cells
to rapidly adapt to new growth environments that may
require one or more new peroxisomal functions. Taken
together, these data stress the exibility of peroxi-
somes; their number, function, and volume fraction is
rapidly adapted to prevailing environmental condi-
tions.
PEROXISOME BIOGENESIS
Genes Involved in Peroxisome Biogenesis
Molecular approaches to identify proteins essential
for peroxisome biogenesis (peroxins) became feasible in
the early 1990s when viable yeast mutants were iso-
lated that were defective in peroxisome development
and function (collectively called pex mutants; see Distel
et al. (1996) for the unied nomenclature). Yeast pex
mutants have lost the capacity to grow on carbon
sources (e.g., fatty acids, methanol) but not nitrogen
sources (e.g., primary amines)that are metabolized
by peroxisomal enzymes. Corresponding genes were
cloned by functional complementation of these mu-
tants, using genomic or cDNA libraries. At present,
23 different PEX genes from various yeasts are now
identied (H. polymorpha, S. cerevisiae, P. pastoris,
and Yarrowia lipolytica; (reviewed by Subramani,
1998; for a recent listing see http://www.peroxisome.
org/). Most PEX gene products (termed peroxins) that
have been identied so far are thought to play a role in
matrix protein import and are discussed below.
Matrix Protein Import Signals
Peroxisomal matrix proteins are encoded by nuclear
genes and are synthesized in the cytosol on free ribo-
somes (Lazarow and Fujiki, 1985). So far, two peroxi-
somal targeting signals have been characterized that
mediate sorting of the protein to peroxisomes (De Hoop
and AB, 1992; Rachubinski and Subramani, 1995; Sub-
ramani, 1998). Most proteins contain a PTS1 signal, a
tripeptide that is located at the extreme C-terminus of
matrix proteins. The PTS1 consensus sequence is
SKL.COOH, but various (conserved) variants of this
motif are allowed (Gould et al., 1989; Lametschwandt-
ner et al., 1998). The PTS2 is located at the N-terminus
and consists of the consensus (R/K)-(L/V/I)-X
5
-(H/Q)-
(L/A) (Swinkels et al., 1991). Most likely other, possibly
internal, signals also exist for a subset of specic pro-
teins, e.g., malate synthase and acyl CoA synthase
(Bruinenberg et al. 1990; Karpichev and Small, 2000;
Kragler et al., 1993; Small et al., 1988). However, the
exact amino acid sequences comprising these signals
have yet to be identied.
Fig. 3. A: Detail of a cell, to demonstrate the temporally matrix
protein import competence of mature peroxisomes. Cells were pre-
grown on methanol/ammonium sulfate, and subsequently switched to
a new growth environment containing methylamine as the sole nitro-
gen source, to induce peroxisomal amine oxidase, key enzyme of
amine metabolism. Two hours after the shift, the cells were analyzed
for the localization of amine oxidase activity (CeCl
3
metylamine).
As evident in the micrograph, only the two small organelles (arrows)
display enzyme activity while staining is absent in the large mature
organelles (glutaraldehyde/OsO
4
). B: Hypothetical model to explain
the temporary matrix protein import capacity of H. polymorpha per-
oxisomes. This model predicts that peroxisomes grow by the uptake of
proteins and lipids. Matrix protein import is facilitated by distinct
protein complexes on the peroxisomal membrane that are donated to
small organelles that bud off from this organelle upon its maturation.
This way, the mature organelle has lost the capacity to incorporate
matrix protein and now serves as an enzyme bag to fulll specic
metabolic functions prescribed by the growth environment. [Color
gure can be viewed in the online issue, which is available at www.
interscience.wiley.com.]
141PEROXISOME ASSEMBLY

PTS1 Protein Import: 1. Receptor
Binding to Matrix Proteins
The initial data on the location of the PTS1-receptor,
Pex5p, in different organisms were conicting and var-
ied with the organisms and/or the experimental ap-
proaches used. The reported locations varied from ex-
clusively associated with the peroxisomal membrane,
solely in the cytosol or the peroxisomal matrix to a dual
location in both the cytosol and the organellar matrix
(Dodt et al., 1995; Elgersma et al., 1996a; Gould et al.,
1996; Szilard et al., 1995; Terlecky et al., 1995; Van der
Klei et al., 1995; Wiemer et al., 1995). This has led to
different models for the mechanisms of PTS1 protein
import. The current view held by most researchers,
except for Y. lipolytica (Szilard et al. 1995), is that the
bulk of Pex5p is present in the cytosol in conjunction
with a minor portion that is associated with peroxi-
somes, bound to the peroxisomal membrane or present
in the organellar matrix (Dodt and Gould, 1996; Sub-
ramani, 1998).
In H. polymorpha, Pex5p is localized in the cytosol
and in the peroxisomal matrix; the putative mem-
brane-bound portion of the protein is invariably below
the limit of detection (Van der Klei et al., 1995, 1998).
These observations have led to our current model that
predicts that H. polymorpha Pex5p functions as a cy-
cling receptor between the cytosol and the peroxisomal
matrix (Van der Klei and Veenhuis, 1996; Fig. 4). This
would implicate that a protein export mechanism must
exist for peroxisomes. Very recently, results have been
presented by Subramani and co-workers that prove
this extended shuttle mechanism for PTS1 protein
import in human peroxisomes (Dammai and Subra-
mani, 2001).
The function of Pex5p in PTS1 import has been stud-
ied in detail. Several authors have shown that the
tetratricopeptide repeats (TPR), localized in the C-ter-
minal two-thirds of Pex5p, bind the PTS1 (Brocard et
al., 1994; Fransen et al., 1995; Szilard and Rachubin-
ski, 2000; Terlecky et al., 1995). In detail, insight in
how the PTS1 signal of a matrix protein can bind the
TPR domains came from the 3-dimensional structure of
the C-terminus of human Pex5p (Gatto et al., 2000) as
well as from mutational analysis of S. cerevisiae Pex5p
(Klein et al., 2001). Furthermore, Lametschwandtner
et al. (1998) showed that additional targeting informa-
tion could be present in the residues preceding the
PTS1 in matrix proteins, which most probably modu-
late the strength of the interaction of the cargo protein
with Pex5p.
PTS1 Protein Import: 2. Receptor-Cargo
Docking to the Peroxisomal Membrane
Upon binding a PTS1, the receptor-cargo complex
interacts at a putative docking site on the peroxisomal
membrane en route to the peroxisomal matrix. This
model requires that the afnity of Pex5p for the dock-
ing site increases upon binding of a PTS1 in order to
prevent competition for the docking site between solu-
ble and cargo-bound Pex5p. Indeed, overexpression of
PEX5 in H. polymorpha cells does not interfere with
PTS1 protein import (van der Klei et al. 1995). Perox-
ins that are believed to participate in Pex5p/cargo
docking include Pex13p, which contains a Src homology
3 (SH3) domain (Barnett et al. 2000; Elgersma et al.,
1996a; Erdmann and Blobel, 1996; Gould et al., 1996),
the coiled-coil protein Pex14p (Albertini et al., 1997;
Brocard et al., 1997; Komori et al., 1997) and Pex17p
(Huhse et al., 1998). Pex5p has been shown to directly
interact with Pex13p (Urquhart et al., 2000) and
Pex14p (Schliebs et al., 1999). Next to this, two hybrid
analysis revealed that Pex14p interacts also with
Pex13p and Pex17p (for reviews see Erdmann et al.,
1997; Subramani, 1998). In the N-terminus of human
Pex5p, a pentapeptide repeat motif (W-x-[E,D,Q,A,S]-
[E,D,Q]-[F,Y]) that is conserved in other Pex5ps was
found to bind to the N-terminus of human Pex14p
(Schliebs et al., 1999). Also in its N-terminus, Pex14p
contains a classical SH3 binding motif, PxxP, to which
the SH3 domain of Pex13p binds. Although Pex5p does
not contain such a PxxP motif, it has an alpha helical
element in its N-terminus that interacts with the SH3
domain of Pex13p in an unconventional, non-PxxP-
related manner (Barnett et al., 2000).
How Pex17p binds Pex14p had not been established
yet. S. cerevisiae Pex17p is a peroxisomal membrane
protein that is involved in matrix protein import (Hu-
hse et al., 1998). Surprisingly, a P. pastoris pex17 mu-
tant also partly mislocalizes peroxisomal membrane
proteins. Furthermore, P. pastoris Pex17p has been
shown not only to interact with Pex14p, but also with
Pex19p, a peroxin that is important for the insertion of
peroxisomal membrane proteins, possibly as a receptor
for the peroxisomal targeting signal of membrane pro-
Fig. 4. Schematic representation of the extended shuttle model for
PTS1 matrix protein import in H. polymorpha. Precurser PTS1 pro-
teins, synthesized in the cytosol, bind to the PTS1 receptor (Pex5p)
and are transported to a peroxisomal docking site, consisting of
Pex13p, Pex14p, and Pex17p. A complex of two ring nger proteins
(Pex10p, Pex12p) that also may contain Pex2p may mediate translo-
cation of the Pex5p.cargo complex. After translocation, dissociation of
the Pex5p.cargo complex takes place, possibly mediated by Pex8p.
Recycling of Pex5p to the cytosol requires the function of at least
Pex4p. Whether the import and export of Pex5p proceeds via the same
gate, which in that case is composed of a large protein complex, or
requires different pores, is totally unknown. [Color gure can be
viewed in the online issue, which is available at www.interscience.
wiley.com.]
142 M. VEENHUIS ET AL.

Citations
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Journal ArticleDOI
TL;DR: This work has combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae and shows that the small GTPase Rho1p is specifically recruited to peroxISomes upon their induction in a process dependent on its interaction with theperoxisome membrane protein Pex25p.
Abstract: 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.

156 citations

Journal ArticleDOI
TL;DR: Like other subcellular organelles, peroxisomes divide and segregate to daughter cells during cell division, but this organelle can also proliferate or be degraded in response to environmental cues.

117 citations


Cites background from "Peroxisome assembly in yeast."

  • ...However, peroxisome number and size can also be induced by peroxisome proliferation, which generally occurs when cells are shifted to nutrients whose metabolism requires peroxisomes and their constituent enzymes [1,13]....

    [...]

  • ...Finally, peroxisomal contents can also vary, as shown for methylotrophic yeasts, in which peroxisomes are populated with enzymes involved in fatty-acid boxidation during growth on oleate, but new peroxisomes are endowed with methanol-assimilation enzymes upon growth on methanol [13]....

    [...]

  • ...First, in constitutively dividing cells, peroxisomes divide, as mitochondria and chloroplasts do, by fission of preexisting peroxisomes, a process we refer to simply as ‘peroxisome division’ [12,13]....

    [...]

  • ...Peroxisome volume changes in response to the import of matrix proteins [13]....

    [...]

Journal ArticleDOI
01 Jan 2007-Genetics
TL;DR: It is proposed that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids.
Abstract: The peroxisome, sole site of β-oxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid Screening of the haploid yeast deletion collection identified ∼130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid Oleate slightly enhances growth of wild-type yeast and inhibits growth of all strains identified by the screen Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cell-wall-associated activities, also prevent oleate toxicity The most oleate-inhibited strains lack Sap190, a putative adaptor for the PP2A-type protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190Δ, sit4Δ, and ilm1Δ strains Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids

107 citations

Journal ArticleDOI
TL;DR: Characteristics of the autophagy of peroxisomes in mammalian cells are discussed and a comprehensive model of their likely mechanism of degradation on the basis of known and common elements from other systems is presented.

103 citations

Journal ArticleDOI
TL;DR: Deletion of the motif from myc-AtPex11e led to peroxisome elongation and fission, indicating that the motif in this isoform promotes fission without elongation.
Abstract: Pex11 homologs and dynamin-related proteins uniquely regulate peroxisome division (cell-cycle-dependent duplication) and proliferation (cell-cycle-independent multiplication). Arabidopsis plants possess five Pex11 homologs designated in this study as AtPex11a, -b, -c, -d and -e. Transcripts for four isoforms were found in Arabidopsis plant parts and in cells in suspension culture; by contrast, AtPex11a transcripts were found only in developing siliques. Within 2.5 hours after biolistic bombardments, myc-tagged or GFP-tagged AtPex11 a, -b, -c, -d and -e individually sorted from the cytosol directly to peroxisomes; none trafficked indirectly through the endoplasmic reticulum. Both termini of myc-tagged AtPex11 b, -c, -d and -e faced the cytosol, whereas the N- and C-termini of myc-AtPex11a faced the cytosol and matrix, respectively. In AtPex11a- or AtPex11e-transformed cells, peroxisomes doubled in number. Those peroxisomes bearing myc-AtPex11a, but not myc-AtPex11e, elongated prior to duplication. In cells transformed with AtPex11c or AtPex11d, peroxisomes elongated without subsequent fission. In AtPex11b-transformed cells, peroxisomes were aggregated and rounded. A C-terminal dilysine motif, present in AtPex11c, -d and -e, was not necessary for AtPex11d-induced peroxisome elongation. However, deletion of the motif from myc-AtPex11e led to peroxisome elongation and fission, indicating that the motif in this isoform promotes fission without elongation. In summary, all five overexpressed AtPex11 isoforms sort directly to peroxisomal membranes where they individually promote duplication (AtPex11a, -e), aggregation (AtPex11b), or elongation without fission (AtPex11c, -d).

102 citations


Cites background from "Peroxisome assembly in yeast."

  • ...) that were at least partly metabolized in peroxisomes (Sakai and Subramani, 2000; Veenhuis et al., 2003)....

    [...]

  • ...Under normal conditions, proliferation of peroxisomes 0.1-0.3 m in diameter was induced in yeasts grown on growth medium substrates (e.g. oleate, methanol, etc.) that were at least partly metabolized in peroxisomes (Sakai and Subramani, 2000; Veenhuis et al., 2003)....

    [...]

References
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1,540 citations


"Peroxisome assembly in yeast." refers background in this paper

  • ...Upon their discovery, peroxisomes were thought to develop by budding from the ER (De Duve and Baudhuin, 1966)....

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Journal ArticleDOI

1,112 citations


"Peroxisome assembly in yeast." refers background in this paper

  • ...…data in yeast provided the first evidence that peroxisomes may multiply by division, a concept that was subsequently substantiated by the finding that peroxisomal proteins are synthesized on free polysomes, followed by post-translational import into peroxisomes (Lazarow and Fujiki, 1985)....

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  • ...Morphological data in yeast provided the first evidence that peroxisomes may multiply by division, a concept that was subsequently substantiated by the finding that peroxisomal proteins are synthesized on free polysomes, followed by post-translational import into peroxisomes (Lazarow and Fujiki, 1985)....

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  • ...Peroxisomal matrix proteins are encoded by nuclear genes and are synthesized in the cytosol on free ribosomes (Lazarow and Fujiki, 1985)....

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Journal ArticleDOI
TL;DR: Results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence.
Abstract: The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the luciferase gene reveals that this peroxisomal targeting signal consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine. When this tripeptide is appended to the COOH terminus of a cytosolic protein (chloramphenicol acetyltransferase), it is sufficient to direct the fusion protein into peroxisomes. Additional mutagenesis experiments reveal that only a limited number of conservative changes can be made in this tripeptide targeting signal without abolishing its activity. These results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence.

1,084 citations


"Peroxisome assembly in yeast." refers background in this paper

  • ...The PTS1 consensus sequence is –SKL.COOH, but various (conserved) variants of this motif are allowed (Gould et al., 1989; Lametschwandtner et al., 1998)....

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Journal ArticleDOI

829 citations


"Peroxisome assembly in yeast." refers background in this paper

  • ...…in such distinct functions as carbon catabolism in fungi, biosynthetic processes (e.g., synthesis of amino acids and penicillin in fungi, and of cholesterol in man), photorespiration in plants, or glycolysis in Trypanosomes (Borst, 1989; Van den Bosch et al., 1992; Veenhuis and Harder, 1988)....

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Journal ArticleDOI
TL;DR: It is concluded that a novel PTS is identified that functions at amino‐terminal or internal locations and is distinct from the C-terminal PTS, which implies the existence of two different routes for targeting proteins into the peroxisomal matrix.
Abstract: 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.

630 citations


"Peroxisome assembly in yeast." refers background in this paper

  • ...The PTS2 is located at the N-terminus and consists of the consensus (R/K)-(L/V/I)-X5-(H/Q)(L/A) (Swinkels et al., 1991)....

    [...]

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