2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1327–1340 doi:10.1242/jcs.157743
RESEARCH ARTICLE
An ancestral role in peroxisome assembly is retained by the
divisional peroxin Pex11 in the yeast Yarrowia lipolytica
ABSTRACT
The peroxin Pex11 has a recognized role in peroxisome division.
Pex11p remodels and elongates peroxisomal membranes prior to
the recruitment of dynamin-related GTPases that act in membrane
scission to divide peroxisomes. We performed a comprehensive
comparative genomics survey to understand the significance of the
evolution of the Pex11 protein family in yeast and other eukaryotes.
Pex11p is highly conserved and ancestral, and has undergone
numerous lineage-specific duplications, whereas other Pex11
protein family members are fungal-specific innovations. Functional
characterization of the in-silico-predicted Pex11 protein family
members of the yeast Yarrowia lipolytica, i.e. Pex11p, Pex11Cp
and Pex11/25p, demonstrated that Pex11Cp and Pex11/25p have a
role in the regulation of peroxisome size and number characteristic
of Pex11 protein family members. Unexpectedly, deletion of PEX11
in Y. lipolytica produces cells that lack morphologically identifiable
peroxisomes, mislocalize peroxisomal matrix proteins and
preferentially degrade peroxisomal membrane proteins, i.e. they
exhibit the classical pex mutant phenotype, which has not been
observed previously in cells deleted for the PEX11 gene. Our
results are consistent with an unprecedented role for Pex11p in de
novo peroxisome assembly.
KEY WORDS: Peroxisome, Pex11, Yarrowia lipolytica, Comparative
genomics, Organelle biogenesis
INTRODUCTION
Peroxisomes are membrane-bounded organelles found in most
eukaryotic cells. They are dynamic and highly responsive to
changing environmental cues. The hallmark metabolic pathways of
peroxisomes are the b-oxidation of fatty acids coupled to the
controlled decomposition of hydrogen peroxide by catalase, but
peroxisomes also exhibit specialized biochemical functions
depending on cell type. The peroxisomes of some eukaryotes
have such highly specialized functions that they are known by
distinctive names (Gabaldón, 2010). Peroxisomes in trypanosomes
are known as glycosomes because they compartmentalize
glycolysis enzymes (Michels et al., 2006), and some plant
peroxisomes are called glyoxysomes because they contain
Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7,
Canada.
*Present address: Section Biomedical Imaging, Department of Diagnostic
Radiology, Christian-Albrechts-University, 24118 Kiel, Germany. `Present
address: Seattle Biomedical Research Institute, Seattle, WA, 98109, USA.
§
These authors contributed equally to this work
"
Author for correspondence ([email protected])
Received 9 June 2014; Accepted 28 January 2015
glyoxylate cycle enzymes (Hayashi et al., 2000). Filamentous
fungi have modified peroxisomes called Woronin bodies involved
in the maintenance of cellular integrity (Liu et al., 2008). The
necessity for functional peroxisomes in human development and
health is evident from the peroxisome biogenesis disorders (PBDs),
a spectrum of fatal diseases in which peroxisomes fail to assemble
correctly (Waterham and Ebberink, 2012; Smith and Aitchison,
2013).
Peroxisomes form through two pathways – growth and division
of existing peroxisomes and de novo biogenesis from the
endoplasmic reticulum (ER). The extent to which one or the other
pathway contributes to peroxisome formation is different for
different cells. In actively growing cells of the yeast Saccharomyces
cerevisiae, peroxisome formation is predominantly by growth and
division, whereas de novo peroxisome formation can occur in the
event of a catastrophic loss of peroxisomes from cells (Motley and
Hettema, 2007). The situation in mammalian cells is less clear, and
studies have concluded that either de novo peroxisome formation or
fission of existing peroxisomes can dominate (Veenhuis and van der
Klei, 2014).
Peroxisome division is achieved by both general organelle
divisional proteins, including the dynamin-related GTPases, and
peroxisome-specific divisional proteins, notably those of the
Pex11 family of peroxins. In S. cerevisiae, the Pex11 family is
composed of Pex11p – the founding member of the family –
Pex25p and Pex27p, whereas in humans, the family is made up of
PEX11a, PEX11b and PEX11c forms (Smith and Aitchison,
2013).
The role of Pex11p in peroxisome division is well established.
Deletion of the PEX11 gene in S. cerevisiae results in cells with
fewer, larger peroxisomes (Erdmann and Blobel, 1995), whereas
overexpression of PEX11 results in cells with an increased
number of smaller peroxisomes or elongated structures that are
thought to be peroxisomes in the process of dividing (Marshall
et al., 1995). Pex11p acts to elongate peroxisomes prior to their
scission and subsequent separation and is proposed to assemble
on the peroxisomal membrane at specific sites, stimulating the
accumulation of phospholipids (Koch et al., 2010). Matrix
proteins can then translocate across the growing tubules, and
Pex11p recruits dynamin-related proteins for membrane scission
(Koch et al., 2010). Pex11p has also been shown to exhibit
membrane remodeling activity in vitro (Opaliński et al., 2011). In
human cells, PEX11c is thought to recruit PEX11a and PEX11b
to the peroxisomal membrane to form PEX11-enriched patches,
leading to peroxisome elongation (Koch et al., 2010). Overall,
these findings support a role for Pex11p in peroxisome division
through membrane remodeling.
Pex11p, Pex25p and Pex27p in S. cerevisiae share sequence
similarity. Likewise, all three proteins share partially redundant
function in peroxisome division; overexpression of PEX11,
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Journal of Cell Science
Jinlan Chang§, Mary J. Klute§, Robert J. Tower*, Fred D. Mast`, Joel B. Dacks and Richard A. Rachubinski"
RESEARCH ARTICLE
Journal of Cell Science (2015) 128, 1327–1340 doi:10.1242/jcs.157743
Fig. 1. Comparative genomic survey of the Pex11 protein family in
Fungi and other eukaryotes. Each column represents a Pex11 family
protein characterized in H. sapiens and/or S. cerevisiae, or identified by a
previous bioinformatic analysis (Kiel et al., 2006). Individual genomes from
the comparative genomics survey are color coded according to eukaryotic
supergroup and grouped according to taxonomic classification. A black circle
indicates the presence of a protein in the indicated taxon based on positive
reciprocal pHMMer searches, gray circles with numbers indicate multiple
paralogs of a protein, and dashes indicate that proteins were not identified in
the indicated taxon. The evolutionary relationships between the genomes
included in this study are given on the left, but the rooting of the tree is
arbitrary. Dotted lines indicate genomes with unknown evolutionary
placement. Asterisks indicate GIM5 homologs. Double asterisks indicate
Pex11p homologs that grouped with Pex11Cp in the phylogenetic analysis.
PEX25 or PEX27 leads to cells with increased numbers of
small peroxisomes, whereas deletion of any of these genes leads
to cells with decreased numbers of enlarged peroxisomes (Smith
et al., 2002; Rottensteiner et al., 2003; Tam et al., 2003).
Bioinformatic analysis identified Pex11Bp, Pex11Cp and Pex11/
25p as putative homologs of Pex11p in fungi (Kiel et al., 2006).
This study raised the interesting possibility that additional
members of the Pex11p family have yet to be identified. We
have now completed the most comprehensive comparative
genomics survey of the Pex11 protein family to date in order to
understand the basis and significance of the conservation and
evolution of the Pex11 protein family in yeast and other
eukaryotes. We show that Pex11p itself is highly conserved and
ancestral, and has undergone numerous lineage-specific
duplications, whereas other Pex11 protein family members are
fungal-specific innovations. We functionally characterized the
bioinformatically predicted Pex11 protein family members
Pex11p, Pex11Cp and Pex11/25p of the yeast Yarrowia
lipolytica and showed a conserved role in the regulation of
peroxisome size and number for Pex11Cp and Pex11/25p. We
also showed that deletion of PEX11 in Y. lipolytica unexpectedly
led to cells that lack morphologically identifiable peroxisomes,
are defective in peroxisomal matrix protein import and show
enhanced degradation of peroxisomal membrane proteins, i.e.
they exhibit the classical pex phenotype, which has not been
observed to date in cells deleted for PEX11. Our results are
consistent with an ancestral role for Pex11p in de novo
peroxisome assembly.
Two studies of PEX gene evolution found that most PEX genes
are of eukaryotic origin and that there are patterns of peroxin
conservation and loss across the lineages of the eukaryotic tree of
life (Gabaldón et al., 2006; Schlüter et al., 2006). Since then, new
PEX genes have been identified (Managadze et al., 2010; Tower
et al., 2011), and the number of eukaryotic genome sequencing
projects has increased dramatically, allowing for a more complete
picture of peroxisomal protein evolution. We therefore revisited
the evolution and distribution of the Pex11 protein family by
completing a comparative genomics survey using 125 genomes
(supplementary material Table S3) spanning the six eukaryotic
supergroups (Walker et al., 2011).
Our analysis showed that Pex11p is conserved throughout the
diversity of eukaryotes, and was likely present at the time of the
last eukaryotic common ancestor (LECA) (Fig. 1). Numerous
genomes contain multiple paralogs of Pex11p; three copies are
present in both Homo sapiens and Trypanosoma brucei – i.e.
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RESULTS
A comparative genomics survey of the Pex11 protein family
Pex11p and the Pex11p-related proteins GIM5A and GIM5B
(Maier et al., 2001) – and five copies are present in the plants
Arabidopsis thaliana (Lingard and Trelease, 2006) and Oryza
sativa (Nayidu et al., 2008). Three copies of Pex11p are present in
the fly Drosophila melanogaster in addition to single paralogs
identified previously (Mast et al., 2011; Faust et al., 2012).
Additional genomes encode multiple copies of Pex11p; there are
two or three copies in Archaeplastida genomes (Chlamydomonas
reinhardtii, Micromonas sp., Ostreococcus tauri, Populus
trichocarpa and Volvox carteri), and five copies in the
rhizarian alga Bigelowiella natans and the ciliate Tetrahymena
thermophila. Peroxisomes have not been reported in many
parasites (de Souza et al., 2004; Gabaldón et al., 2006;
Gabaldón, 2010), and it was therefore not unexpected that
Pex11p was not identified in the parasite genomes analyzed in
this study – Encephalitozoon cuniculi, Entamoeba histolytica,
Giardia sp., Trichomonas vaginalis, Theileria parva and
Plasmodium falciparum. Pex11p was identified in the parasite
Toxoplasma gondii, although peroxisomes have not been
observed in this organism (Ding et al., 2000). Failure to
identify Pex11p homologs in the remaining eukaryotic genomes
could be because an organism lacks peroxisomes but has never
been experimentally characterized, or the genomes encode highly
divergent sequences that were not detected by our search
algorithms, or because of poor genome assembly and/or coverage.
Unlike for most parasites, a divergent peroxisome called the
glycosome has been reported for the Kinetoplastida (GualdrónLópez et al., 2012). The trypanosome genomes encode multiple
Pex11 proteins, with Pex11p itself or GIM5 as the top reciprocal
pHMMer hits in the T. brucei genome (Fig. 1). An evolutionary
relationship was proposed to exist between GIM5 and
trypanosome Pex11p on the basis of sequence similarity and
common function (Voncken et al., 2003). T. brucei GIM5A and
GIM5B share 13% and 14% sequence identity with T. brucei
Pex11p (Voncken et al., 2003). Knockdown of GIM5 expression
resulted in decreased numbers of larger glycosomes, whereas
overexpression of GIM5 resulted in increased numbers of smaller
glycosomes (Maier et al., 2001; Voncken et al., 2003). However,
we did not find strong support for kinetoplastid Pex11p and GIM5
proteins being homologous, as reciprocal pHMMer searches into
kinetoplastid genomes retrieved only the query sequence.
Although GIM5 proteins could still be true Pex11p homologs,
they are highly divergent in sequence and frequently failed to
retrieve Pex11p homologs in non-kinetoplastid genomes (data not
shown). A phylogenetic analysis of the Pex11 family proteins in
Excavata resolved Pex11p and GIM5 into distinct clades,
predating the split of kinetoplastids from their relative, Bodo
saltans (supplementary material Fig. S1).
With few exceptions, the fungal genomes in this study all
encoded one Pex11p ortholog. Similarly, Pex11Cp was identified
across all lineages of Fungi studied, but less frequently in other
supergroups. However, paralogs of the remaining Pex11 family
proteins were observed in Fungi. Pex27p, initially reported in S.
cerevisiae, was identified only in the Saccharomyces species S.
paradoxes and S. bayanus (Fig. 1). Pex25p homologs were
restricted to the Saccharomycotina, a lineage of ascomycete fungi
(James et al., 2006). Pex11Bp, Pex11Cp and Pex11/25p are
present in many more fungal genomes than just the filamentous
Fungi, as initially reported (Kiel et al., 2006). Pex11Bp homologs
were identified in members of the Pezizomycotina, which is a
collective of fungal lineages more basal to the Saccharomycotina
(James et al., 2006). Pex11/25p homologs, with the exception of a
Journal of Cell Science (2015) 128, 1327–1340 doi:10.1242/jcs.157743
Pex11/25p homolog identified in Y. lipolytica, were restricted to
more basal fungal lineages outside the Ascomycota. These
findings indicate that all Pex11 family proteins, with the
exception of Pex11p itself, appear to be present only in the
Fungi, and their distribution might have been sculpted by fungal
evolution.
Evolution of the Pex11 protein family
The Pex11 protein family has undergone multiple expansions in
diverse eukaryotic lineages that, by and large, are not observed
for the other peroxins (Smith and Aitchison, 2013). Our
comparative genomics survey confirmed that S. cerevisiae
Pex25p and Pex27p are homologs, as a pHMMer search into
the S. cerevisiae genome with Pex25p as the query retrieved
Pex25p but also Pex27p as the next-best hit (E-value55.3610210),
whereas a pHMMer search with Pex27p as query retrieved
Pex27p but also Pex25p as the next-best hit (E-value5
5.6610212). We also investigated the specific evolutionary
relationship between Pex25p and Pex27p, and Pex11p. A Pex11p
hidden Markov model (HMM) retrieved S. cerevisiae Pex25p and
Pex27p, in addition to Pex11p. Similarly, a Pex25p HMM retrieved
Pex11p in some Fungi (data not shown). A Pex27p HMM did not
retrieve Pex11p, probably because Pex27p is restricted to the genus
Saccharomyces.
Are the proposed Pex11 family proteins Pex11Bp, Pex11Cp and
Pex11/25p genuine Pex11p homologs? HMMer searches with
Pex11Bp or Pex11Cp as queries retrieved Pex11p homologs in
yeast, in addition to Pex11Bp or Pex11Cp homologs (data not
shown). Likewise, HMMer searches with Pex11p queries retrieved
some yeast Pex11Bp and Pex11Cp homologs, as well as Pex11p
homologs (data not shown). The identities of some proteins
identified in our comparative genomics survey were sometimes
ambiguous. For example, Chlamydomonas reinhardtii, Micromonas
sp., Volvox carteri, Bigelowiella natans, Tetrahymena thermophila
and Naegleria gruberi proteins were retrieved by both Pex11p and
Pex11Bp HMMs (supplementary material Table S1). A Pex11Cp
HMM retrieved proteins not only in Nematostella vectensis,
Bigelowiella natans, Ectocarpus siliculosus, Phytophthora
ramorum and Guillardia theta, but also retrieved Pex11c in H.
sapiens, Mus musculus, Canis familiaris and Danio rerio. Pex11/
25p was originally identified in Y. lipolytica as a protein with weak
similarity to both Pex11p and Pex25p (Kiel et al., 2006); however,
our alignments do not show evidence of a clear fusion protein (data
not shown). Searches with a Pex11p HMM in the Y. lipolytica
genome retrieved the original Y. lipolytica Pex11/25p query in
addition to Pex11p. pHMMer searches with Pex11p and Pex11/25p
queries occasionally retrieved the same yeast proteins (data not
shown).
Clearly, the evolution of the Pex11 protein family is quite
complex. We therefore undertook a phylogenetic analysis to
elucidate further the evolutionary histories of the Pex11 family
proteins in the Opisthokonta supergroup and to clarify the
classification of some homologs whose identities were ambiguous
based on reciprocal pHMMer searches alone (Fig. 2). A strongly
supported clade comprising mammalian Pex11c and fungal
Pex11Cp homologs was observed (Fig. 2). Strikingly, this
suggests that these proteins are specific orthologs of the same
gene and the products of a gene duplication event occurring in at
least the ancestor of opisthokonts. This analysis resolved each of
the fungal Pex11 family proteins into well supported clades.
Pex11Bp, identified in Pezizomycotina fungi, arose within the
Pex11p clade and appears to be a PEX11 gene duplication in
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these fungal lineages. Pex27p arose in the Pex25p clade; PEX25
and PEX27 have been identified as ohnologs arising from a
Saccharomyces whole genome duplication (Byrne and Wolfe,
2005). Based on the comparative genomic and phylogenetic
analyses, the conservation and broad distribution of Pex11p
identifies it as the ancestral member of the Pex11 protein family
and shows that other members of this family, aside from
Pex11Cp, are fungal-specific innovations.
Y. lipolytica Pex11 protein family members are peroxisomal
integral membrane proteins
This and earlier (Kiel et al., 2006; Schlüter et al., 2006; Gabaldón
et al., 2006) comparative genomics surveys and phylogenetic
analyses have elucidated the evolution of the Pex11 protein
family in Fungi and other eukaryotes. In Y. lipolytica, all
members of the Pex11 protein family have been predicted only in
silico, and nothing is known regarding their localization in the
cell and whether they have a role in peroxisome biogenesis. We
therefore chose to functionally characterize the Pex11 protein
family, i.e. Pex11p, Pex11Cp and Pex11/25p, in Y. lipolytica. Y.
lipolytica shares characteristics with both the pezizomycete and
the saccharomycete yeasts, and its genome has been shown
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previously to be taxonomically informative for understanding the
evolution of Rab GTPases in yeasts (Pereira-Leal, 2008).
We first determined whether the Y. lipolytica Pex11 protein
family members are peroxisomal (Fig. 3A). We tagged Pex11p,
Pex11Cp and Pex11/25p at their C-termini with mCherry (mC)
and visualized their localization by confocal microscopy.
Pex11p–mC, Pex11Cp–mC and Pex11/25p–mC colocalized
with the GFP-tagged peroxisomal matrix protein thiolase
(Pot1p–GFP) to punctate structures characteristic of peroxisomes.
Subcellular fractionation showed that Pex11p–mC, Pex11Cp–mC
and Pex11/25p–mC localized preferentially to a 20,000 g pellet
(20KgP) fraction enriched for peroxisomes and not to a supernatant
fraction (20KgS) (Fig. 3B). Density gradient centrifugation of the
20KgP fraction showed that these three proteins cofractionate with
peroxisomal Pot1p but not with mitochondrial Sdh2p (Fig. 3C).
These data show that Pex11p, Pex11Cp and Pex11/25p are
peroxisomal in Y. lipolytica.
We next determined whether Pex11p, Pex11Cp and Pex11/25p
associate with the peroxisomal membrane. Pex11p is predicted to
have two transmembrane helices (amino acids 103–122 and 137–
156), but no transmembrane domains were predicted for Pex11Cp
or Pex11/25p (http://www.cbs.dtu.dk/services/TMHMM-2.0/).
Journal of Cell Science
Fig. 2. Phylogenetic analysis of Pex11 family proteins in Opisthokonta. Node values indicate statistical support by MrBayes/PhyML/RAxML (posterior
probability/bootstrap value/bootstrap value), with statistical values for highly supported nodes replaced by symbols as indicated. Best Bayesian topology is
shown rooted on the O. tauri and C. merolae Pex11p sequences as outgroups. Species names are color coded according to Fig. 1. A supported Holozoa
Pex11c and fungal Pex11Cp clade is observed. Fungal Pex11/25p/Pex25p/Pex27p, Holozoa Pex11a/Pex11b and fungal Pex11p/Pex11Bp are also resolved into
separate clades.
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Journal of Cell Science (2015) 128, 1327–1340 doi:10.1242/jcs.157743
The 20KgP fraction was subjected to hypotonic lysis in dilute
alkali Tris buffer followed by centrifugation (Fig. 3D). Pex11p–
mC, Pex11Cp–mC and Pex11/25p–mC localized preferentially to
the pellet fraction (Ti8P) enriched for membrane proteins like the
peroxisomal integral membrane protein Pex2p (Eitzen et al.,
1996) and not to the supernatant fraction (Ti8S) enriched for
matrix proteins like Pot1p. Ti8P fractions were next subjected
to extraction with alkali sodium bicarbonate followed by
centrifugation. Pex11p–mC, Pex11Cp–mC and Pex11/25p–mC
localized preferentially to the pellet fraction (CO3P) enriched for
integral membrane proteins, as did Pex2p, in contrast to Pot1p
(Fig. 3D). Taken together, these observations indicate that
Pex11p, Pex11Cp and Pex11/25p are peroxisomal integral
membrane proteins.
Transcript and protein levels of PEX11 gene family members
PEX11 and PEX25 increase, whereas those of family member
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Fig. 3. Y. lipolytica Pex11p, Pex11Cp and Pex11/
25p are peroxisomal integral membrane proteins.
(A) Pex11p–mC, Pex11Cp–mC and Pex11/25p–mC
colocalize with the GFP-tagged peroxisomal matrix
enzyme thiolase (Pot1p–GFP) as shown by confocal
microscopy. Scale bar: 5 mm. (B) Pex11p–mC,
Pex11Cp–mC and Pex11/25p–mC localize
preferentially to the 20KgP fraction enriched for
peroxisomes. Equivalent proportions of fractions were
analyzed by immunoblotting using antibodies against
Pot1p and against DsRed to detect mCherry-tagged
proteins. (C) Pex11p–mC, Pex11Cp–mC and Pex11/
25p–mC cofractionate with peroxisomal Pot1p.
Organelles in the 20KgP fraction were subjected to
density gradient centrifugation, and fractions were
collected from the bottom of the gradient. Equivalent
volumes of fractions were analyzed by immunoblotting
using antibodies against DsRed to detect mCherrytagged proteins. Fractions enriched for peroxisomes
and mitochondria were identified using antibodies
against Pot1p and Sdh2p, respectively. (D) Pex11p,
Pex11Cp and Pex11/25p are peroxisomal integral
membrane proteins. The 20KgP fraction from
cells expressing Pex11p–mC, Pex11Cp–mC or Pex11/
25p–mC was treated with dilute alkali Tris buffer to
rupture peroxisomes and subjected to centrifugation to
yield a supernatant (Ti8S) fraction enriched for matrix
proteins and a pellet (Ti8P) fraction enriched for
membrane proteins. Ti8P fractions were further treated
with alkali Na2CO3 and separated by centrifugation into
a supernatant (CO3S) fraction enriched for peripherally
associated membrane proteins and a pellet (CO3P)
fraction enriched for integral membrane proteins.
Pex11p–mC, Pex11Cp–mC and Pex11/25p–mC all
enriched in the Ti8P and CO3P fractions, as did the
known peroxisomal integral membrane protein Pex2p.
Pot1p marks the fractionation profile of a peroxisomal
matrix protein. Equivalent proportions of fractions were
analyzed by immunoblotting with antibodies against
DsRed, Pex2p and Pot1p. (E) Transcription of the
genes PEX11, PEX11C and PEX11/25 is induced by
incubation of Y. lipolytica in oleic acid medium. Wildtype cells grown in glucose-containing YEPD medium
were transferred to, and incubated in, oleic-acidcontaining YPBO medium. Aliquots of cells were
collected at the times indicated, and total RNA was
isolated and subjected to semi-quantitative RT-PCR
analysis. RT-PCR products were resolved by
electrophoresis on 2% agarose, detected by staining
with ethidium bromide and quantified using ImageJ
software. Ratios of the staining intensities of RT-PCR
products from the genes indicated and from the internal
control gene, G6PDH, were calculated and normalized
to the ratio obtained at 0 h for each of the genes, which
was set at 1. Quantitative data show the mean6s.e.m.
(four independent experiments).
PEX27 remain constant, in S. cerevisiae during incubation in
oleic acid medium (Erdmann and Blobel, 1995; Smith et al.,
2002; Tam et al., 2003). Semi-quantitative RT-PCR was
performed to determine transcript levels of the PEX11,
PEX11C and PEX11/25 genes in Y. lipolytica during a switch
from glucose medium to oleic acid medium (Fig. 3E).
Quantification showed that the transcript levels of PEX11,
PEX11C and PEX11/25 increased with time of incubation in
oleic acid medium, whereas transcript levels for the cytosolic
enzyme G6PDH remained constant and served as an internal
control (Fig. 3E).
Peroxisomes are absent in pex11D cells, and are larger and
fewer in number in pex11CD and pex11/25D cells
Deletion of genes like PEX3 encoding a peroxin essential for the
formation of the peroxisomal membrane leads to abnormal
peroxisome assembly and failure of yeast strains to grow on
medium containing oleic acid, a carbon source whose metabolism
requires functional peroxisomes (Fig. 4A). Growth of pex11CD
and pex11/25D strains on oleic acid medium was similar to that of
the wild-type strain. In contrast, the pex11D strain, like the pex3D
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strain, displayed no growth on oleic acid medium, and thus
Pex11p does not share redundant function with Pex11Cp or
Pex11/25p. Our findings also suggest that Y. lipolytica pex11D
cells lack functional peroxisomes, in contrast to what has been
observed for other eukaryotic cells lacking functional Pex11p,
which contain fewer and enlarged peroxisomes, prompting
further investigation into a possible role for Pex11p in
peroxisome assembly in Y. lipolytica.
We next examined peroxisome morphology in the pex11D
strain, as the inability to grow on oleic acid medium is often
indicative of defective peroxisome assembly. Deletion of PEX11
in cells expressing genomically encoded Pot1p–GFP and incubated
in oleic acid medium showed fluorescently labeled tendrillar
structures (supplementary material Fig. S2A), However, the
presence of elongated peroxisomes in these pex11D cells was not
supported by electron microscopy (Fig. 5A), biochemical analysis
(Fig. 6A) or immunofluorescence analysis (supplementary
material Fig. S2B) of the pex11D strain. Moreover, pex3D
cells expressing genomically encoded Pot1p–GFP also showed
a similar fluorescent elongated and tendrillar pattern in oleic
acid medium (supplementary material Fig. S2A); however,
Fig. 4. Peroxisomes are absent in pex11D cells and fewer in number in pex11CD and pex11/25D cells. (A) pex11D cells, but not pex11CD or pex11/25D
cells, display a growth defect on oleic acid medium. Cells of the wild-type strain and the designated deletion strains were grown in glucose-containing YEPD,
spotted onto oleic-acid-containing YPBO agar at tenfold serial dilutions and an initial OD600 of 1, and grown for 2 days at 28˚C. (B) Wild-type and pex11D
cells containing plasmid expressing Pot1p–mRFP (red) were grown in YEPD-hygromycin B (YEPD) or grown in YEPD-hygromycin B and then transferred to
YPBO-hygromycin B for 8 h (YPBO). The periphery of cells is shown in blue. Representative images are shown. (C) Wild-type, pex11CD and pex11/25D cells
expressing genomically integrated POT1-GFP were grown in YEPD to log phase and imaged by confocal microscopy (YEPD) or grown in YEPD, transferred to
YPBO, grown to log phase and imaged (YPBO). Representative images are shown. Scale bars: 5 mm. The number of peroxisomes (Pot1p–GFP-containing
puncta) per mm2 was determined with Imaris software. Graphs display the results of three independent experiments for each strain. The data show the
mean6s.e.m. Significance was determined by using Student’s t-test with respect to the wild-type strain. N.S., not significant; **P,0.01.
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pex3D cells of Y. lipolytica failed to assemble functional
peroxisomes and showed a general mislocalization of
peroxisomal matrix proteins to the cytosol (Bascom et al.,
2003). We speculate that the elongated fluorescent structures
observed in pex11D and pex3D cells expressing genomically
integrated POT1-GFP are an artifact of oligomerization of
Pot1p–GFP that is mislocalized to the cytosol. Confocal
microscopy showed that monomeric Pot1p–mRFP expressed
from plasmid in pex11D cells showed a generalized
fluorescence pattern characteristic of the cytosol and lacked
the readily identifiable punctate peroxisomes still observed in
wild-type cells (Fig. 4B).
We also examined whether deletion of PEX11C or PEX11/25
affected peroxisome dynamics, particularly peroxisome division,
which is well documented for deletions of genes encoding other
Pex11 protein family members. We used confocal microscopy to
observe wild-type, pex11CD and pex11/25D cells expressing
genomically encoded Pot1p–GFP in both glucose medium and
oleic acid medium. Cells of the pex11CD and pex11/25D strains
in glucose-containing YEPD showed no significant difference in
peroxisome number compared to wild-type cells by confocal
microscopy (Fig. 4C). In contrast, pex11CD and pex11/25D cells
in oleic-acid-containing YPBO contained fewer peroxisomes
compared with wild-type cells (Fig. 4C).
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Fig. 5. pex11D cells contain numerous small
vesicles, whereas pex11CD and pex11/25D cells
contain enlarged peroxisomes. (A) Ultrastructure of
pex11D cells. Wild-type peroxisomal profiles (B) are
absent in pex11D cells. Insets present enlarged views
of boxed areas showing increased numbers of small
vesicles in pex11D cells. (B) Ultrastructure and
morphometric analysis of wild-type, pex11CD and
pex11/25D cells. Cells were grown in YEPD,
transferred to YPBO for 8 h and processed for electron
microscopy. Representative images are shown. P,
peroxisome. Scale bars: 1 mm. The areas of individual
peroxisomes of 100 randomly selected cells were
morphometrically analyzed using Olympus iTEM
software. Peroxisomes were then separated into size
categories. A histogram was generated for each strain
depicting the percentage of total peroxisomes
occupied by peroxisomes of each category. The
numbers along the x-axis are the maximum sizes of
peroxisomes (mm2) in each category, except for the
last value, which represents the minimum size of
peroxisomes (mm2) in the last category. (C) The
estimated number of peroxisomes per cubic
micrometer of cells of the wild-type strain and of the
mutant strains pex11CD and pex11/25D. (D) Average
peroxisome size (mm2) for cells of the wild-type strain
and of the mutant strains pex11CD and pex11/25D.
The data show the mean6s.e.m. The significance of
the comparison of the average peroxisome area of
each deletion strain with that of the wild-type strain
was determined by using Student’s t-test
. ***P,0.001.
Fig. 6. Deletion of PEX11 results in abnormal localization of
peroxisomal matrix proteins and increased degradation of peroxisomal
membrane proteins. (A) Cells of the wild-type strain and deletion strains
pex11D, pex11CD and pex11/25D were subjected to subcellular fractionation
to yield a 20KgS fraction and a 20KgP fraction enriched for peroxisomes. The
20KgS fraction was subjected to additional centrifugation to yield a
200KgS fraction enriched for cytosol and a 200KgP fraction enriched for
small vesicles. Equivalent volumes of all fractions were analyzed by
immunoblotting. Anti-Aox3p antibodies recognize subunits Aox2p, Aox3p
and Aox4p of fatty acyl-CoA oxidase. (B) Deletion of PEX11 prevents
proteolytic processing of Pot1p to its mature form. Whole-cell lysates of the
wild-type strain and deletion strains pex11D, pex11CD and pex11/25D were
subjected to immunoblotting with antibodies against Pot1p. mPot1p,
mature form of Pot1p; pPot1p, precursor form of Pot1p.
Electron microscopy showed canonical spherical peroxisomes in
wild-type cells (Fig. 5B) but no evidence of wild-type peroxisomes
in pex11D cells (Fig. 5A). However, clusters of small vesicles were
observed in some pex11D cells (Fig. 5A, boxed regions and insets).
These findings, together with evidence from confocal microscopy
(Fig. 4B; supplementary material Fig. S2B), show that pex11D
cells fail to assemble morphologically identifiable, functional
peroxisomes. Morphometric analysis revealed that pex11CD and
pex11/25D cells contained fewer and larger peroxisomes compared
with wild-type cells (Fig. 5B–D).
pex11D cells exhibit abnormal localization of peroxisomal
matrix proteins and enhanced degradation of peroxisomal
membrane proteins
Unlike other eukaryotes investigated so far, Y. lipolytica cells
deleted for PEX11 do not contain morphologically identifiable
peroxisomes. These unexpected findings prompted us to examine
the localization of peroxisomal matrix and membrane proteins in
this strain. Cells of the wild-type strain and of the deletion strains
pex11D, pex11CD and pex11/25D were subjected to differential
centrifugation to yield a 20KgS fraction and a 20KgP fraction
enriched for ‘mature’ peroxisomes (Titorenko and Rachubinski,
2000; Titorenko et al., 2000). The 20KgS fraction was subjected
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Journal of Cell Science (2015) 128, 1327–1340 doi:10.1242/jcs.157743
to ultracentrifugation at 200,000 g to yield a pellet (200KgP)
fraction enriched for small vesicles, including small peroxisomal
vesicles (Titorenko and Rachubinski, 2000; Titorenko et al.,
2000) and a supernatant (200KgS) fraction enriched for cytosol.
Immunoblotting was performed with antibodies against matrix
proteins with different peroxisomal targeting signals (PTSs): the
PTS1-containing protein isocitrate lyase (ICL), a 62-kDa protein
recognized by antibodies against the tripeptide Ser-Lys-Leu
(SKL) PTS1 consensus sequence, the PTS2-containing protein
Pot1p and five acyl-CoA oxidase subunits (Aox1–5) that contain
neither a PTS1 nor a PTS2 sequence. These matrix proteins
localized preferentially to the 20KgP fraction from wild-type,
pex11CD and pex11/25D cells but, in contrast, were detected
predominately in the 20KgS and 200KgS fractions from pex11D
cells (Fig. 6A). These findings indicate that pex11D cells are
defective in the import of peroxisomal matrix proteins, regardless
of their PTS type.
Like matrix proteins, the peroxisomal membrane proteins
Pex2p, Pex3Bp and Pex24p were enriched in the 20KgP fraction
from wild-type, pex11CD and pex11/25D cells (Fig. 6A).
Surprisingly, Pex2p and Pex24p were not detected in subcellular
fractions from pex11D cells, whereas a small amount of Pex3Bp
was found in the 200KgP fraction containing small vesicles. These
data are consistent with a preferential degradation of peroxisomal
membrane proteins in the absence of ‘mature’ peroxisomes in
pex11D cells and are further supportive of pex11D cells being
defective in peroxisome assembly.
In wild-type cells, Pot1p is imported into the peroxisomal
matrix as a 45-kDa precursor form (pPot1p), where it is
proteolytically processed to its mature 43-kDa form (mPot1p)
(Titorenko and Rachubinski, 1998). Only mPot1p was detected in
the lysates of wild-type, pex11CD and pex11/25D cells by
immunoblot; however, only pPot1p was detected in the pex11D
lysate (Fig. 6B), further supporting that pex11D cells are
defective in matrix protein import.
PEX11, but not PEX11C or PEX11/25, exhibits differential
control of peroxisome size and number under different
growth conditions
We next examined the effect of overexpressing PEX11, PEX11C
or PEX11/25 on peroxisome size and number. Electron
microscopy analysis showed that cells overexpressing PEX11C
or PEX11/25, but not PEX11, contained increased numbers of
smaller peroxisomes in oleic acid medium (Fig. 7A–C). In
glucose medium, cells overexpressing PEX11, PEX11C or
PEX11/25 all contained increased numbers of peroxisomes
(supplementary material Fig. S3). Taken together, the data from
our gene deletion and overexpression studies demonstrate a
classical Pex11 protein family member role in maintenance of
peroxisome size and number for Pex11Cp and Pex11/25p, but
also unexpectedly show that Pex11p in Y. lipolytica functions
differentially in controlling peroxisome size and number under
different carbon conditions and, more notably, functions as a
bona fide peroxisome biogenic protein, akin to Pex1p or Pex14p
for example (Smith and Aitchison, 2013).
PEX11 from Hansenula polymorpha and Pichia pastoris, but
not S. cerevisiae PEX11 or Y. lipolytica PEX11C or PEX11/25,
can complement the peroxisome assembly defect of Y.
lipolytica pex11D cells
Deletion of the PEX11 gene led to the inability of Y. lipolytica
to grow on oleic-acid-containing YPBO agar (Fig. 4A).
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Fig. 7. Overexpression of PEX11C or PEX11/25, but not PEX11, results in increased numbers of smaller peroxisomes in cells incubated in oleic acid
medium. (A) Ultrastructure and morphometric analysis of cells overexpressing (OE) PEX11, PEX11C or PEX11/25, and a control strain containing the parental
vector pTC3. Cells were grown in glucose medium, transferred to oleic acid medium, incubated for 20 h and processed for electron microscopy.
Representative images are shown. P, peroxisome. Scale bar: 1 mm. Morphometric analysis was performed as described in the legend for Fig. 5B. (B) The
estimated number of peroxisomes per cubic micrometer of cells of the wild-type strain and of the mutant strains pex11CD and pex11/25D. (C) Average
peroxisome size (mm2) for cells of the wild-type strain and of the mutant strains pex11CD and pex11/25D. The data show the mean6s.e.m. The significance of
the comparison of the average peroxisome area of each deletion strain with that of the wild-type strain was determined by using Student’s t-test. N.S., not
significant; ***P,0.001.
Y. lipolytica Pex11p that has been lost in S. cerevisiae Pex11p and
is absent in Y. lipolytica Pex11Cp or Pex11/25p (Fig. 2).
Pex11p, Pex11Cp and Pex11/25p are mislocalized in pex3D
and pex19D cells
Our results show that Y. lipolytica pex11D cells do not assemble
functional peroxisomes, do not contain morphologically
identifiable peroxisomes, mislocalize peroxisomal matrix
proteins and more readily degrade peroxisomal membrane
proteins, all of which support an unprecedented role for the
divisional protein Pex11 in de novo peroxisome assembly.
Peroxisomes are linked to the cellular endomembrane
trafficking system by the ER. Peroxisomal membrane proteins
sample the ER en route to peroxisomes, and peroxisome growth
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Transformation of the Y. lipolytica pex11D strain with plasmid
expressing Y. lipolytica PEX11 re-established the growth of the
strain on YPBO (Fig. 8), showing that the failure of pex11D cells
to grow on oleic acid medium was due to the absence of the Y.
lipolytica PEX11 gene. We next tested whether Hansenula
polymorpha Pex11p, Pichia pastoris Pex11p, S. cerevisiae
Pex11p and Y. lipolytica Pex11Cp or Pex11/25p could correct
the aberrant peroxisome assembly in Y. lipoytica pex11D cells
and restore their ability to grow on oleic acid medium. Expression
of plasmid-borne H. polymorpha and P. pastoris PEX11, but not
S. cerevisiae PEX11 or Y. lipolytica PEX11C or PEX11/25,
restored growth of the Y. lipolytica pex11D strain on oleic acid
medium (Fig. 8), suggesting that H. polymorpha and P. pastoris
Pex11p have retained a peroxisome biogenic function present in
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Journal of Cell Science (2015) 128, 1327–1340 doi:10.1242/jcs.157743
Fig. 8. Expression of PEX11 from H. polymorpha and P. pastoris, but not expression of S. cerevisiae PEX11 or Y. lipolytica PEX11C or PEX11/25,
restores the ability to grow on oleic acid medium to Y. lipolytica pex11D cells. (A) Y. lipolytica pex11D cells were transformed with empty vector pUB4 or
pUB4 encoding H. polymorpha PEX11 (HpPEX11), P. pastoris PEX11 (PpPEX11), S. cerevisiae PEX11 (ScPEX11) or Y. lipolytica PEX11 (YlPEX11),
grown in YEPD-hygromycin B, spotted onto YEPD-hygromycin B or YPBO-hygromycin B agar at tenfold serial dilutions and an initial OD600 of 1, and grown for
1 day at 28˚C. (B) Y. lipolytica pex11D cells were transformed with pUB4 or pUB4 encoding Y. lipolytica PEX11, PEX11C or PEX11/25, grown in YEPDhygromycin B, spotted onto YPBO-hygromycin B agar at tenfold serial dilutions and an initial OD600 of 1, and grown for 2 days at 28˚C.
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membranes of the secretory system that do not assemble into
functional peroxisomes.
DISCUSSION
Limited functional analysis has been performed on the novel insilico-predicted Pex11p-related proteins in Fungi (Kiel et al.,
2006). The Pex11 protein family in the fungus Penicillium
chrysogenum was recently characterized, and Pex11p and
Pex11Cp were found to be peroxisomal, but Pex11Bp was
localized to the ER (Opaliński et al., 2012). Deletion of PEX11B
or PEX11C had no significant effect on peroxisome number,
whereas overexpression of PEX11B resulted in clusters of smaller
peroxisomes. The authors concluded that Pex11Bp might play
some role in peroxisome biogenesis, although not in de novo
peroxisome assembly. The Pex11 protein family in the yeast H.
polymorpha was also recently investigated, and Pex11p, Pex11Cp
and Pex25p were shown to be peroxisomal (Saraya et al., 2011).
Intriguingly, H. polymorpha Pex25p, like S. cerevisiae Pex25p,
was shown to be required for de novo peroxisome biogenesis, as
the presence of Pex25p, but not Pex11p, was needed to
reintroduce peroxisomes into a peroxisome-deficient strain.
We have now demonstrated that Pex11Cp and Pex11/25p
regulate peroxisome size and number. In this way, Pex11Cp and
Journal of Cell Science
and division are facilitated by proteins and lipids coming from the
ER. The functions of the peroxins Pex3p and Pex19p in these
processes are well documented in different eukaryotes (Smith
and Aitchison, 2013; Tabak et al., 2013). In Y. lipolytica, pex3D
cells lack any peroxisomal structures (Bascom et al., 2003)
and pex19D cells, although they do contain morphologically
identifiable peroxisomes, mislocalize peroxisomal matrix
proteins to the cytosol (Lambkin and Rachubinski, 2001). We
assessed the localization of Pex11p–mC, Pex11Cp–mC and
Pex11/25p–mC in pex3D and pex19D cells by confocal
microscopy. Distinctive ‘rosette’ structures decorated by
Pex11p–mC were observed in pex3D and pex19D cells grown
in either YEPD or YPBO (supplementary material Fig. S4A,
boxed regions and insets). Pex11Cp–mC in both pex3D and
pex19D cells exhibited a distinctive cortical and perinuclear
localization reminiscent of the ER (supplementary material Fig.
S4A) and similar in appearance to the pattern exhibited by the
ER-localized marker protein, GFP–HDEL (supplementary
material Fig. S4B). In contrast, Pex11/25p–mC exhibited a
generalized cortical localization in pex3D and pex19D cells
(supplementary material Fig. S4A). Our observations suggest that
in pex3D and pex19D cells, Pex11p, Pex11Cp and Pex11/25p are
localized to membrane structures and, in the case of Pex11Cp, to
Pex11/25p function similarly to Pex11 family proteins in other
eukaryotes. Y. lipolytica pex11CD or pex11/25D cells grown in
glucose medium do not contain significantly different numbers of
peroxisomes from wild-type cells, whereas pex11CD and pex11/
25D strains grown in oleic acid medium contain fewer
peroxisomes than wild-type cells. These observations suggest
that Pex11Cp and Pex11/25p in Y. lipolytica might act to regulate
peroxisome size and number in a proliferating rather than
constitutively dividing peroxisome population.
Pex11 family proteins are associated with the peroxisomal
membrane, but their specific membrane topology varies with the
organism (Schrader et al., 2012). S. cerevisiae Pex11p, Pex25p
and Pex27p appear to be peripherally associated with the
peroxisomal membrane, although some controversy still exists
regarding the strength of the association between Pex11p and the
membrane. Mammalian Pex11 proteins are integral membrane
proteins with one or two transmembrane-spanning helices. We
showed that Y. lipolytica Pex11p is a peroxisomal integral
membrane protein (Fig. 3D), with two membrane-spanning
domains predicted. The membrane topology of Y. lipolytica
Pex11p therefore more closely resembles its human homologs
than S. cerevisiae Pex11p. Organelle extraction also demonstrated
that Pex11Cp and Pex11/25p are integral to the peroxisomal
membrane (Fig. 3D). Although transmembrane domains were
not predicted for Pex11Cp or Pex11/25p, these proteins contain
regions of hydrophobicity that might promote integral association
with the peroxisomal membrane.
pex3D and pex19D cells of Y. lipolytica are defective in
peroxisome biogenesis and lack wild-type peroxisomes (Lambkin
and Rachubinski, 2001; Bascom et al., 2003). Here, we showed
that Pex11p is also implicated in peroxisome biogenesis in Y.
lipolytica and demonstrated a putative localization of Pex11
family proteins to compartments of the secretory system in
pex3D and pex19D strains (supplementary material Fig. S4A,B).
Pex11p–mC was found in distinctive rosette structures in pex3D
and pex19D cells (supplementary material Fig. S4A). Pex11p
could be modifying the ER membrane in these strains; however,
we did not observe evidence of modified ER in electron
micrographs of pex11D cells. The specific nature of these
structures awaits further investigation.
Why does deletion of Y. lipolytica PEX11 alone produce a
defect in peroxisome assembly resulting in the absence of
peroxisomes? One explanation could be the nature of peroxisome
dynamics in Y. lipolytica, particularly in comparison to those in S.
cerevisiae. Yeast peroxisomes increase in size and number when
grown in medium containing a non-fermentable carbon course.
One key reason why Y. lipolytica is such a tractable model to
study peroxisome biogenesis is because of its ability to efficiently
utilize hydrophobic substrates such as oleic acid, which is
accompanied by extensive proliferation of peroxisomes.
However, growth of S. cerevisiae in oleic acid medium has
been shown to result primarily in increased peroxisomal size
rather than increased peroxisomal number (Tower et al., 2011). In
contrast, Y. lipolytica cells contain greatly enlarged and much
more numerous peroxisomes when grown in oleic acid medium as
compared to growth in glucose medium (Smith and Rachubinski,
2001; Chang et al., 2009; 2013). The increased number of
peroxisomes in Y. lipolytica cells, coupled with the
responsiveness of this yeast to the presence of fatty acid with
regards to its increased capacity to proliferate peroxisomes, could
explain why a defect in peroxisome assembly was not heretofore
observed in other yeasts deleted for PEX11. The loss of Pex11p in
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Y. lipolytica could result in the uncoupling of the process of de
novo peroxisome biogenesis from the process of peroxisome
growth and division. This uncoupling could, in turn, lead to
dysregulation of peroxisome biogenesis and loss of the organelle,
evidence for which is observed in our inability to detect several
peroxisomal membrane proteins by immunoblotting of subcellular
fractions of pex11D cells (Fig. 6A).
Another reason why a role in peroxisome assembly per se had
not been before attributed to the Pex11 protein is that peroxisome
assembly, rather than peroxisome division, was an ancestral
function of the Pex11 protein and this function has been retained
by other members of the Pex11 protein family. For instance,
although humans have three Pex11 proteins, only mutation of
PEX11b leads to mislocalization of a matrix protein and
pathology associated with the PBDs (Ebberink et al., 2012).
Similarly, a role in de novo peroxisome biogenesis has been
attributed to Pex25p in S. cerevisiae and H. polymorpha (Smith
et al., 2002; Saraya et al., 2011). Our phylogenetic analysis
demonstrated that Pex25p did not arise by duplication of the gene
encoding Pex11p at the Y. lipolytica branch point and resolved
the Pex11 family of proteins in Fungi into at least three wellsupported clades (Pex11p/Pex11Bp, Pex11Cp, Pex11/25p/
Pex25p/Pex27p) (Fig. 2). These data point to multiple
expansions of the Pex11 family, giving rise to the various
Pex11 proteins in Fungi. More specifically, given that both
Pex11p and Pex25p have been implicated in peroxisome
biogenesis, our analysis suggests that there were multiple
ancestral Pex11 sequences present in the ancestor of Fungi.
One of these sequences had a role in peroxisome biogenesis, and
different paralogs have retained this ancestral peroxisome
biogenic function. The ability of H. polymorpha and P. pastoris
PEX11, but not S. cerevisiae PEX11, to correct the growth defect
of the Y. lipolytica pex11D strain on oleic acid medium (Fig. 8),
the metabolism of which requires functional peroxisomes,
suggests that H. polymorpha and P. pastoris Pex11 proteins
have retained a peroxisome biogenic function that is present in Y.
lipolytica Pex11p and that has been lost in S. cerevisiae Pex11p.
Pex11 family proteins can form homo-oligomeric and heterooligomeric protein complexes (Schrader et al., 2012), and
constructive neutral evolution supports the retention of gene
duplication events within such complexes. Thus, while there is
one set of evolutionary pressures supporting the expansion of the
PEX11 family, which is possibly independent of the function of
the Pex11 family protein complex, taxon-specific functional
specialization could selectively retain the separate functions of
the complex within distinct paralogs or divide the task among all
members in an idiosyncratic manner. This hypothesis is supported
by different lines of functional evidence in Fungi, as no single
Pex11 family protein is essential for peroxisome biogenesis in P.
chrysogenum (Opaliński et al., 2012), whereas the ancestral role
of PEX11 in peroxisome biogenesis has been retained by PEX11
in Y. lipolytica, and perhaps by H. polymorpha and P. pastoris
PEX11, as well as by PEX25 in other yeasts (Smith et al., 2002;
Saraya et al., 2011).
Unlike most Pex proteins, the Pex11 protein family has
undergone multiple expansions. Our analysis resolved mammalian
Pex11a/Pex11b and Pex11c into separate clades, pre-dating the
Opisthokonta, or earlier (Fig. 2). The distribution observed for
the Pex11 protein family and its roles in peroxisomal dynamics
are indicative of a protein family that has undergone multiple
independent gene duplications and losses repeatedly through diverse
eukaryotic lineages.
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In closing, we have shown a role for the classical divisional
peroxin Pex11p in peroxisome assembly in the yeast Y. lipolytica.
We have also provided insight into novel members of the Pex11
protein family in Fungi by demonstrating that Pex11Cp and
Pex11/25p play a conserved role in the regulation of peroxisome
size and number. By undertaking an evolutionary cell biological
study on the history of a protein family and studying peroxisomes
in diverse eukaryotes, we were able to uncover a new and
unexpected role for Pex11p in de novo peroxisome assembly,
with implications for human health through increased
understanding of a new class of PBD arising from mutation of
a gene encoding a Pex11 protein isoform.
Journal of Cell Science (2015) 128, 1327–1340 doi:10.1242/jcs.157743
v.8.0.0 (Stamatakis, 2006) were used for maximum likelihood analyses,
with bootstrap values based on 100 pseudoreplicates of each
dataset. RAxML trees were run using CIPRES (http://www.phylo.org/
index.php).
Strains and culture conditions
Y. lipolytica strains are listed in supplementary material Table S2. Strains
were cultured at 30 ˚C unless otherwise indicated. Plasmid-containing
strains were cultured in synthetic minimal medium or in YEPD medium
or YPBO medium supplemented with hygromycin B (125 mg/ml) or
nourseothricin (100 mg/ml). Synthetic minimal medium consisted of
0.67% yeast nitrogen base without amino acids, 2% glucose, 16complete
supplement mixture without leucine or uracil. YEPD, YPBO, YEPA and
YNO media have been described previously (Chang et al., 2013).
MATERIALS AND METHODS
Comparative genomics
Integrative transformation of yeast
Genomes surveyed in this study are listed in supplementary material
Table S3. To identify putative Pex11p, Pex11/25p, Pex25p and Pex27p
homologs, H. sapiens, S. cerevisiae and/or Y. lipolytica protein sequences
(supplementary material Table S1) were used as queries for pHMMer
(Eddy, 2009) searches against locally hosted genomes. Candidate
homologs with an expect (E-) value less than or equal to 0.05 were
subjected to reciprocal pHMMer searches against the query genome and
the non-redundant genome. Retrieval of the original query as the top
reciprocal pHMMer hit with an E-value less than or equal to 0.05 and
retrieval of a named homolog as the top reciprocal pHMMer hit in the
non-redundant database were the criteria for the identification of
homologs. Verified homologs were aligned using MUSCLE v.3.6
(Edgar, 2004). Hidden Markov models (HMM) (Eddy, 1998) were
built from the alignments and used by HMMer 3.0 (Eddy, 2009) for
searches against locally hosted genomes. Candidate homologs with Evalues less than or equal to 0.05 were verified as described above.
Validated homologs from the same eukaryotic supergroup and/or
experimentally characterized homologs from the literature were used as
queries for pHMMer searches. Newly validated homologs were added to
the HMM iteratively, and HMMer searches were repeated until no
additional putative homologs were identified. For genomes without
protein sequences, nHMMer (Wheeler and Eddy, 2013) was used to
search nucleotide databases following the procedure described above.
To identify putative Pex11Bp and Pex11Cp homologs, protein
sequences for known Pex11Bp and Pex11Cp homologs (Kiel et al.,
2006) (supplementary material Table S1) were aligned using MUSCLE
3.6. HMMs were built from the alignments and used by HMMer 3.0 to
search locally hosted genomes. Candidate homologs were verified as
described above. Newly validated homologs were added to the HMM
iteratively, and HMMer searches were repeated until no additional
putative homologs were identified. For genomes without protein
sequences, HMMs were used by nHMMer to search nucleotide
databases following the procedure described above.
Evolutionary relationships between taxa (Fig. 1) were determined
using http://www.ncbi.nlm.nih.gov/taxonomy (Aime et al., 2006;
Hibbett, 2006; James et al., 2006; Suh et al., 2006; Wapinski et al.,
2007; Boehm et al., 2009; Schoch et al., 2009; Medina et al., 2011;
Walker et al., 2011; Ebersberger et al., 2012; Mast et al., 2012).
Y. lipolytica PEX11 (XP_501425.1), PEX11C (XP_501447.1) and
PEX11/25 (XP_503276.1) were disrupted using a fusion PCR-based
integrative procedure (Davidson et al., 2002).
Phylogenetics
Subcellular fractionation, and isolation and extraction
of peroxisomes
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pTC3 (Lin et al., 1999), pUB4 (Kerscher et al., 2001), pINA443 (Nuttley
et al., 1993) and pINA445 (Nuttley et al., 1993) have been described
previously. pTC3-NAT was constructed from pTC3 by replacing URA3
with the Streptomyces nouresi NAT gene conferring resistance to
nourseothricin. The chimeric genes PEX11-mCherry and PEX11/25mCherry flanked by promoter and terminator regions of the POT1 gene
were inserted into pUB4. PEX11C-mCherry flanked by promoter and
terminator regions of POT1 was cloned into pTC3-NAT. H. polymorpha
PEX11, P. pastoris PEX11, S. cerevisiae PEX11 and Y. lipolytica PEX11,
PEX11C and PEX11/25 flanked by promoter and terminator regions of
POT1 were inserted into pUB4. The gene encoding the ER chimeric
marker protein GFP–HDEL was inserted into pINA445. pUB4-mRFPSKL and pUB4-POT1-mRFP have been described previously (Chang
et al., 2009; 2013).
Microscopy
Confocal fluorescence microscopy (Tower et al., 2011), electron
microscopy and morphometric analysis (Eitzen et al., 1997; Tam et al.,
2003), and immunofluorescence microscopy (Tam and Rachubinski,
2002) were performed as described previously.
Deconvolution, image manipulation and quantification of
peroxisome number
Images were deconvolved using Huygens Professional Software
(Scientific Volume Imaging BV). Three-dimensional (3D) data sets
were deconvolved using an iterative Classic Maximum Likelihood
Estimation confocal algorithm with an experimentally derived point
spread function. Imaris software (Bitplane) was used to create maximum
intensity projections of the deconvolved 3D datasets. Transmission
images of yeast cells were processed by applying a Gaussian filter in
Huygens, and blue color was applied to images using Imaris software.
Internal structures in images were removed in Photoshop to better
visualize fluorescence data.
Wild-type cells expressing Pex11p–mC, Pex11Cp–mC or Pex11/25p–mC
were grown in glucose medium and transferred to oleic acid medium for
8 h. Subcellular fractionation to yield a postnuclear supernatant (PNS)
fraction, a 20KgP pellet fraction enriched for peroxisomes, a 20KgS
supernatant fraction enriched for cytosol, a 200KgP pellet fraction
enriched for small vesicles and a 200KgS cytosolic supernatant fraction
was performed as described previously (Tam and Rachubinski, 2002;
Chang et al., 2009).
Peroxisomes were purified by isopycnic centrifugation on a
discontinuous Nycodenz gradient as described previously (Chang et al.,
2009). Extraction of peroxisomes with dilute alkali Tris to yield a pellet
Journal of Cell Science
MUSCLE v.3.6 was used to align validated homologs. ZORRO (Wu
et al., 2012) was used to calculate HMM scores for each position in the
alignment. Alignments (available upon request) were masked and
trimmed in Mesquite v.2.75 (www.mesquiteproject.org). ProtTest v.1.3
(Abascal et al., 2005) was used to determine the optimal model of
sequence evolution. MrBayes v.3.2 (Ronquist and Huelsenbeck, 2003)
was used for Bayesian analysis to produce posterior probability values.
Analyses were run for 1,000,000 Markov chain Monte Carlo generations.
Two independent runs were performed, with convergence of the results
confirmed by ensuring a Splits Frequency of ,0.1. Burn-in values were
obtained by removing all trees prior to a graphically determined plateau.
Additionally, PhyML v.2.44 (Guindon and Gascuel, 2003) and RAxML
Plasmids
(Ti8P) fraction enriched for membrane proteins and a supernatant (Ti8S)
fraction enriched for matrix proteins was performed as described
previously (Vizeacoumar et al., 2003). Extraction of the Ti8P fraction
with alkali Na2CO3 and centrifugation to yield a pellet (CO3P) fraction
enriched for integral membrane proteins and a supernatant (CO3S)
fraction enriched for peripheral membrane proteins were performed as
described previously (Chang et al., 2009).
Semi-quantitative RT-PCR analysis
Total RNA was isolated using the RiboPure Yeast Kit (Life
Technologies). First-strand cDNA was synthesized using SuperScript
VILO MasterMix (Life Technologies).
Antibodies
Antibodies have been described previously (Tam and Rachubinski, 2002;
Chang et al., 2009). Rabbit anti-DsRed polyclonal antibody was from
Takara Bio. Alexa-Fluor-488-conjugated anti-guinea-pig-IgG secondary
antibody was from Life Technologies. Antigen–antibody complexes were
detected by enhanced chemiluminescence.
Acknowledgements
We thank Emily Herman for writing scripts used in the comparative genomics
survey and for helpful discussion; Hanna Kroliczak, Richard Poirier, Elena
Savidov and Nasser Tahbaz for expert technical assistance; and members of the
Rachubinski and Dacks laboratories for helpful discussion. We dedicate this
manuscript to the memory of our good friend and colleague, Richard Poirier.
Competing interests
The authors declare no competing or financial interests.
Author contributions
J.C., M.J.K., J.B.D. and R.A.R. provided a conceptual framework for the study,
interpreted data and wrote the manuscript. J.C., M.J.K., R.J.T. and F.D.M.
performed the experiments.
Funding
M.J.K. is the recipient of a Faculty of Medicine and Dentistry/Alberta Health
Services Graduate Student Recruitment Studentship; and a Queen Elizabeth II
Graduate Scholarship. J.B.D. is the Canada Research Chair in Evolutionary Cell
Biology. This work was supported by the Canadian Institutes of Health Research
[grant number 53326 to R.A.R.]; and a New Faculty Award from Alberta Innovates
Technology Futures to J.B.D.
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.157743/-/DC1
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