A Comprehensive Overview of the Vertebrate p24 Family: Identification of
a Novel Tissue-Specifically Expressed Member
Jeroen R.P.M. Strating,*1 Nick H.M. van Bakel,* Jack A.M. Leunissen, and Gerard J.M. Martens*
*Department of Molecular Animal Physiology, Donders Centre for Neuroscience and Nijmegen Centre for Molecular Life Sciences
(NCMLS), Faculty of Science, Radboud University, Nijmegen, The Netherlands; and Laboratory of Bioinformatics, Wageningen
University, Wageningen, The Netherlands
The members of the p24 protein family have an important but unclear role in transport processes in the early secretory
pathway. The p24 family consists of four subfamilies (a, b, c, and d), whereby the exact composition of the family varies
among species. Despite more than 15 years of p24 research, the vertebrate p24 family is still surprisingly ill
characterized. Here, we describe the human, mouse, Xenopus, and zebrafish orthologues of 10 p24 family members and
a new member that we term p24c5. Of these eleven p24 family members, nine are conserved throughout the vertebrate
lineage, whereas two (p24c4 and p24d2) occur in some but not all vertebrates. We further show that all p24 proteins are
widely expressed in mouse, except for p24a1 and p24c5 that display restricted expression patterns. Thus, we present for
the first time a comprehensive overview of the phylogeny and expression of the vertebrate p24 protein family.
Introduction
The secretory pathway comprises a number of distinct
membrane-bounded organelles involved in the transport,
maturation, and sorting of biologically active proteins destined for the plasma membrane or the extracellular space.
Proteins enter the secretory pathway in the endoplasmic reticulum (ER) where they are properly folded. They are then
transported to the Golgi from where they are targeted to their
final intra- or extracellular destination (Palade 1975). The
members of the p24 protein family play an important role
in vesicular transport processes at the ER–Golgi interface,
although their exact function is still unclear (roles suggested
for p24 have been reviewed in Emery et al. 1999; Kaiser
2000; Kuiper and Martens 2000; Carney and Bowen 2004).
The p24 proteins constitute a family of type-I transmembrane (TM) proteins of ;24 kDa. The family can be
subdivided into four subfamilies termed a, b, c, and d
(Dominguez et al. 1998). The p24 proteins share a similar
architecture that includes a single TM helix. At the lumenal
side, the p24 proteins have an N-terminal Golgi dynamics
(GOLD) domain that may play a role in the incorporation
of cargo into transport vesicles (Anantharaman and Aravind
2002). In the structure of some family members, this domain
is followed by a putative coiled-coil region that may interact
with other p24 proteins (Ciufo and Boyd 2000). At the cytoplasmic side, the p24 proteins have a short C-terminal tail
of 12–22 amino acids (aas) that contains binding motifs for
the vesicle coat complexes COPI and COPII (Fiedler et al.
1996; Sohn et al. 1996; Dominguez et al. 1998; Harter and
Wieland 1998; Belden and Barlowe 2001; Béthune et al.
2006).
Despite its crucial role in the cell, a comprehensive
overview of the vertebrate p24 family is not available
and the occurrence of orthologues of the various p24 family
members among vertebrate species is surprisingly ill documented, as are their tissue distributions. In contrast, the
1
Present address: Heidelberg University Biochemistry Center
(BZH), Im Neuenheimer Feld 328, Heidelberg, Germany.
Key words: early secretory pathway, phylogeny, evolutionary tree,
tissue distribution, p23, p25, p26, p27, tp24, gp25L, TMED.
E-mail: [email protected].
Mol. Biol. Evol. 26(8):1707–1714. 2009
doi:10.1093/molbev/msp099
Advance Access publication May 8, 2009
Ó The Author 2009. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
compositions of the p24 families in yeast and fruit fly,
and their expression patterns in fly developmental stages
and tissues have been extensively studied (Schimmöller
et al. 1995; Belden and Barlowe 1996; Marzioch et al.
1999; Carney and Taylor 2003; Bartoszewski et al.
2004; Boltz et al. 2007). In this paper, we describe the composition in vertebrates and evolutionary aspects of the p24
family as well as the tissue distributions of the mouse p24
family members.
Materials and Methods
Bioinformatic Methods
Searches against the nonredundant protein database at
the National Center for Biotechnology Information (NCBI)
were performed using the BlastP algorithm. Genomic databases at the NCBI (March 2008 releases) were queried using the TBlastN algorithm. Sequences were aligned using
ClustalW 1.83 (Thompson et al. 1994) and MUSCLE (Edgar 2004), and edited using JalView 2.3 (Clamp et al. 2004;
Waterhouse et al. 2009). The alignments were colored for
conservation (threshold 25%) using the ClustalX coloring
algorithm (Thompson et al. 1997). A detailed description of
this coloring algorithm is available through the ClustalX
documentation (available on www.clustal.org). In short,
this algorithm assigns colors to aligned positions based
on the type of aa residue in the consensus and the conservation of its physicochemical properties. The intensity of
the coloring (as set by the threshold) is determined by
the degree of conservation of the consensus residue type.
Phylogenetic trees were built using PHYLIP 3.67, PAUP
4.0b10, and MrBayes 3.1.1, and plotted with DRAWTREE
(PHYLIP package; fig. 1) and SVGTree (Leunissen JAM,
unpublished data; supplementary figs. 2 and 3, Supplementary Material online). The phylogenetic trees calculated by
the various programs were in general agreement with each
other, apart from minor variations in branch lengths and
bootstrap values. Signal peptides were predicted using
the public SignalP 3.0 server (Bendtsen et al. 2004). Generally, the Neural networks and the Hidden Markov models
algorithms yielded the same result. If not, the predicted signal peptide cleavage position was taken that was most consistent with that of the most closely related p24 proteins.
TM helices were predicted using the public TMHMM
1708 Strating et al.
FIG. 1.—Unrooted tree of the p24 protein family. A multiple alignment of p24 protein sequences was performed (supplementary fig. 1,
Supplementary Material online) and an unrooted cladogram tree was generated. The four subfamilies are indicated by gray background shading. Hs,
Homo sapiens; Pt, Pan troglodytes; Mm, Mus musculus; Xt, Xenopus tropicalis; Dr, Danio rerio; Tn, Tetraodon nigroviridis; Dm, Drosophila
melanogaster; Sc, Saccharomyces cerevisiae; and At, Arabidopsis thaliana.
2.0 server (Krogh et al. 2001). In general, the predicted TM
helices within different p24 proteins aligned well.
RNA Isolation and Reverse Transcriptase (RT)-PCR
Female BALB/c surplus mice (12 months old) were
sacrificed by cervical dislocation in accordance with institutional guidelines, and tissues were dissected and snap frozen
in liquid nitrogen. Total RNA was extracted from the tissues
using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA concentrations were determined
spectrophotometrically and checked for integrity of the
rRNAs by agarose gel electrophoresis. The Trizol method
yielded high-quality RNA from all tissues, except for pancreas. In order to isolate high-quality RNA from pancreas
suitable for RT-PCR, we used the RNAlater ice solution
(Ambion) but again we were not successful, probably be-
cause of the abundant presence of pancreatic RNases. We
therefore could not examine the mRNA expression of the
mouse p24 proteins in pancreas. The RNA samples were
treated with DNaseI (Fermentas) and cDNA was generated
using a RevertAid H-minus first strand cDNA synthesis kit
(Fermentas) according to the manufacturer’s protocol. Standard PCR reactions were performed using intron-spanning
primer pairs (supplementary table 1, Supplementary Material
online). A PCR for b-actin was performed as a control for
equal input.
Results
p24 Nomenclature
In the past, various nomenclatures for vertebrate p24
proteins have been introduced and are still being used in
The Vertebrate p24 Family 1709
Table 1
Nomenclature and Occurrence of the Vertebrate p24 Proteins
a
b
c
d
Systematic Name
Systematic Database Namea
Alternative Names
Mammals
Amphibians
Fish
p24a1
p24a2
p24a3
p24b1
p24c1
p24c2
p24c3
p24c4
p24c5
p24d1
p24d2
TMED11
TMED9
TMED4
TMED2
TMED1
TMED5
TMED7
TMED3
TMED6
TMED10
gp25L
p25, GMP25, gp25L2, p24d, p24D
GMP25iso
p24, p24a
tp24, T1/ST2 (receptor-binding protein)c
p28, T1/ST2 isoc
(g)p27
p26, p24b
þb
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þd
þ
þ
þ
þe
þ
p23, tmp21(I), p24c
p23iso
a
In the protein database, most p24 proteins are annotated as ‘‘TMED’’ (TM emp24 protein transport domain containing; emp24 is the first yeast p24 protein identified;
Schimmöller et al. 1995). The nonredundant NCBI protein database contains one protein (TMED8) that is erroneously annotated as being related to the p24 proteins, because
of its size (;300 aas) and the fact that it lacks a TM domain and the two absolutely conserved cysteine residues (see text). The TMED8 protein contains a GOLD domain in
its C-terminal region. A Blast search for TMED8-related proteins retrieved several GOLD domain-containing proteins, indicating that TMED8 is a GOLD domaincontaining protein but not a member of the p24 family.
b
The human p24a1 gene contains an in-frame stop codon and therefore the p24a1 protein does not appear to occur in humans. However, other primates have an intact
p24a1 (see text).
c
p24c1 is sometimes erroneously termed T1/ST2, instead of the original name T1/ST2 receptor binding protein (Gayle et al. 1996), and because of its close
relationship p24c2 is sometimes—also erroneously—named T1/ST2 iso.
d
Zebrafish contains two p24c1 inparalogues (see text).
e
The present assembly of the zebrafish genome does not contain a full-length p24c5 sequence, but the Tetraodon nigroviridis genome does (see text).
parallel (table 1). Work on mammalian p24 proteins has
mostly focused on six family members, usually termed
p23, p24, p25, p26, p27, and tp24. A more systematic approach, involving the use of a Greek letter (to identify a subfamily of structurally related p24 proteins) followed by
a number (starting with the first-discovered member), has
been proposed by Dominguez et al. (1998). Here, we use this
systematic nomenclature because it provides direct information as to which subfamily the p24 protein belongs and the
subfamily can be expanded relatively easy with a newly
identified family member (based on its structure). A careful
literature search revealed the occurrence of 10 p24 proteins in
vertebrate species, namely, gp25L/p24a1 (Wada et al. 1991),
p25/p24a2 (Dominguez et al. 1998), GMP25iso/p24a3
(Rötter et al. 2002), p24/p24b1 (Stamnes et al. 1995), tp24/
p24c1 (Gayle et al. 1996), p28/p24c2 (Rötter et al. 2002),
p27/p24c3 (Dominguez et al. 1998), p26/p24c4 (Dominguez
et al. 1998), p23/p24d1 (Sohn et al. 1996), and p23iso/p24d2
(Holthuis et al. 1995; Kuiper et al. 2000) (table 1). In invertebrates, yet other nomenclatures exist (e.g., see Marzioch
et al. 1999 for an overview of the p24 proteins in yeast and
Boltz et al. 2007 for the Drosophila p24 proteins).
The Vertebrate p24 Proteins
To provide a comprehensive overview of the p24 family in vertebrates, we chose to compare the p24 protein sequences from human and vertebrate model organisms,
namely, mouse (Mus musculus), amphibian (Xenopus tropicalis), and zebrafish (Danio rerio). The genome-sequencing
projects for these species have either been completed or are
in a far-advanced stage, which enables the identification of
any novel, unannotated members of the p24 family. We retrieved the known p24 proteins from the protein database
using a text-based search and the unannotated family members with a Blast search against the nonredundant protein
database or the respective genome and expressed sequence
tag (EST) databases. All p24 proteins have orthologues in
all vertebrate species (table 1), except for the few cases discussed below. In addition, we identified a novel member of
the p24 family that we termed p24c5 (see below). From
table 1, it is clear that most vertebrate genomes encode
10 p24 family members.
The Human p24a1 Open Reading Frame (ORF) Contains
a Premature Stop Codon
The gp25L/p24a1 protein, originally found in dog
pancreatic microsomes, was the first member of the p24
family to be identified (Wada et al. 1991). Interestingly,
we found during our searches that the human sequence
for p24a1 contains an in-frame stop codon (codon 111;
TAA). This stop codon was found in both the reference
and the alternate human genome assembly and in human
ESTs for p24a1. A stop codon in the ORF of p24a1 was
found only in human, but not in other vertebrates. In most
other vertebrates (e.g., orangutan, chimp, macaque, bushbaby, mouse lemur, cow, cat, mouse, and rat) codon 111 is
TCA (encoding a serine), whereas in some other species
(e.g., horse, dog, opossum, and microbat), it is TCC or
TCG (also encoding a serine). The chimpanzee p24a1
ORF is identical to the human p24a1 sequence, except
for 3 nt changes, one of which corresponds to the A/C difference in codon 111 (the two other differences are silent).
On the basis of the available data, we conclude that humans lack an intact p24a1 protein. Therefore, we decided
to include chimpanzee p24a1 in our phylogenetic analysis
as a representative of primate p24a1.
Zebrafish Has Two p24c1 Copies
In the database, we found two zebrafish protein sequences corresponding to p24c1, which we termed p24c1A and
p24c1B. The p24c1A protein is encoded by a gene on
1710 Strating et al.
chromosome 3, whereas the p24c1B protein is derived from
a gene on a 29.90-kb scaffold (Scaffold Zv7_NA1556) that
has yet to be assigned to a genomic location. The lengths of
the zebrafish p24c1A and p24c1B proteins are identical (226
aa residues), whereby the two protein sequences are 61.5%
identical and 77.4% similar (data not shown). A comparative
aa sequence analysis with other p24 family members from
various vertebrate species clearly grouped both sequences
with human, mouse, and Xenopus p24c1 (fig. 1 and supplementary fig. 2, Supplementary Material online). This indicates that the two proteins are derived from two different
p24c1 genes rather than from splicing isoforms or different
alleles of the same gene. Thus, the two zebrafish p24c1 proteins most likely are inparalogues (Sonnhammer and Koonin
2002) resulting from a genome duplication event early in the
teleost lineage (Jaillon et al. 2004).
Amphibians Lack a p24c4 Orthologue but Have an
Additional Amphibian-Specific p24d2
Despite extensive searches in genome and EST databases of both X. tropicalis and Xenopus laevis, we did not
identify a Xenopus orthologue of p24c4. Because all other
vertebrates included in our analyses have p24c4 orthologues, Xenopus likely lost this family member during evolution. With respect to the p24d subfamily, it is interesting
to note that we previously found two subfamily members in
X. laevis (p24d1 and p24d2; Holthuis et al. 1995; Kuiper
et al. 2000). Because we did not identify an orthologue
of Xenopus p24d2 in any other vertebrate branch, we conclude that p24d2 is an amphibian-specific member of the
p24 family. Interestingly, a second expressed p24d gene
(Tmp21-II) has been identified in human (Blum et al.
1996), but this gene does not encode a complete p24 protein
due to a change in reading frame and a nonsense mutation
(Horer et al. 1999). At the protein sequence level, human
Tmp21-II grouped with human and mouse p24d1 (data not
shown). Therefore, it is not an orthologue of Xenopus p24d2
but rather a nonfunctional inparalogue of p24d1. An orthologue of Tmp21-II occurs in chimp (with a stop codon located slightly further upstream than in the human sequence)
but not in mouse or rat, suggesting that the duplication
event took place recently in the primate radiation.
A Novel Member of the Vertebrate p24 Family: p24c5
During our Blast searches for orthologues of known
p24 proteins, we serendipitously found a novel protein
(TMED6) that based on sequence similarity appeared to
be a member of the p24 family. Because in our phylogenetic
analysis this protein belongs to the p24c subfamily, we
termed this novel family member p24c5. We next identified
p24c5 orthologues in a large number of evolutionarily distant vertebrate species (human, mouse, cow, dog, platypus,
opossum, Xenopus, and pufferfish). The zebrafish genome
assembly used in our searches (Zv7) contained only two
fragments of the p24c5 sequence (located on different scaffolds that both mapped to chromosome 25), but not a fulllength sequence. Because we identified the complete
protein sequence of a p24c5 orthologue in another teleost
fish, the green-spotted pufferfish Tetraodon nigroviridis,
we chose to include this sequence as a representative of fish
p24c5 in our phylogenetic analyses.
Phylogenetic Relationships between the p24 Proteins
After having retrieved the orthologues of all known
p24 proteins and the novel family member p24c5, we used
all mouse p24 protein sequences to query the mouse genome for any other novel family member, but we did
not identify any additional p24 sequences. In addition to
the protein sequences of human, mouse, Xenopus, and zebrafish, we chose to include in our phylogenetic analyses
the model invertebrate Drosophila melanogaster (fruit
fly), the model yeast Saccharomyces cerevisiae (budding
yeast), and the model plant Arabidopsis thaliana. Fruit
fly and yeast have representatives of all four p24 subfamilies (Marzioch et al. 1999; Boltz et al. 2007), whereas plants
contain only members of the p24b and p24d subfamilies
(Carney and Bowen 2004).
Alignment of the various p24 protein sequences
showed that overall these proteins display a low degree
of aa sequence identity (supplementary fig. 1, Supplementary Material online). In all p24 proteins, only the two cysteine residues of the GOLD domain are fully conserved
(Anantharaman and Aravind 2002). A number of other
aa residues show a high degree of sequence identity or similarity, in particular in the TM region, for example, a glutamine residue that has been shown to be involved in the
trafficking of p24 proteins (Fiedler and Rothman 1997).
Next, we predicted the positions of the TM helices in all
p24 proteins included in our analysis. In general, the predicted TM helices could be easily aligned (positions 250–
272 in supplementary fig. 1, Supplementary Material online; TM length of 23 residues). Of particular interest is that
the predicted TM domains in all p24a proteins are three aa
residues shorter (positions 250–269; a length of 20 residues) than those of the p24b, -c and -d proteins. The cytoplasmic tails of most p24 proteins contain an FF-motif
involved in COPII binding (Dominguez et al. 1998) or
an alternative motif consisting of other aromatic or large
hydrophobic residues (supplementary fig. 1, Supplementary Material online). In addition, the majority of the p24
proteins has a doublet of basic aas located two positions
C-terminal of the FF, together yielding the FFXXBBXn
motif (n 2; B indicates any basic residue) involved in
COPI binding (Béthune et al. 2006). Within each of the four
vertebrate p24 subfamilies, the degree of aa sequence identity between the various members of that particular subfamily is high (supplementary fig. 3, Supplementary Material
online). Furthermore, with the exception of the c-subfamily,
all vertebrate p24 subfamilies have extremely wellconserved cytoplasmic tails that provide ‘‘signatures’’ to
identify a particular subfamily.
Figure 1 (an unrooted cladogram tree) and supplementary figure 2, Supplementary Material online (a bootstrapped midpoint-rooted tree) show the relationships between
the various p24 proteins and subfamilies. The phylogenetic
relationships among the orthologuous p24 proteins correspond to the evolutionary relationships of the species from
which they are derived, with the exception of the p24d
branch. In the phylogenetic trees, zebrafish p24d1 appears
The Vertebrate p24 Family 1711
more closely related to mouse and human p24d1 than Xenopus p24d1 (fig. 1 and supplementary figs. 2 and 3B, Supplementary Material online). However, the bootstrap values
for the branching of Xenopus and zebrafish p24d1 are relatively low (supplementary figs. 2 and 3B, Supplementary
Material online), indicating that the p24d1 proteins are too
closely related to reliably calculate their phylogenetic descent. Alternatively, zebrafish p24d1 has undergone a number of reverting mutations that cause it to resemble the
mammalian p24d1 proteins more than Xenopus p24d1.
It appears that the p24a and p24d subfamilies share
a common ancestor, as do the p24b and p24c subfamilies.
The four p24 subfamilies branch off very early and are present in yeast and all animals. We therefore conclude that the
common ancestor to fungi and animals likely contained
members of all p24 subfamilies. Interestingly, although
the plant p24 proteins group in only two subfamilies
(p24b and p24d), plants contain descendants of both the
p24a/d and the p24b/c ancestors. Assuming that the root
of the tree (fig. 1 and supplementary fig. 2, Supplementary
Material online) lies in between the a/d and the b/c stems
implicates that the a/d and b/c ancestors were already present in the common ancestor to plants and animals. This
again implies that a loss of two genes in plants is more likely
than the gain of two genes in animals/fungi. Furthermore,
subfamily expansion appears to have taken place relatively
recently in evolution and independently in vertebrates and
other species (fig. 1 and supplementary fig. 2, Supplementary Material online). For example, the yeast p24a subfamily forms a separate group from the vertebrate a subfamily,
although both seem to have a single common p24a ancestor. An even clearer example may be provided by the plant
p24d subfamily that seems to have greatly expanded independently from the fungi/animals from a single p24d ancestor. Interestingly, all vertebrate p24 orthologues are
evolutionarily well conserved. In particular, the p24b1 protein is extremely well conserved (fig. 1, supplementary figs.
1—3, Supplementary Material online), for example, the
p24b1 proteins from the distantly related species man
and zebrafish show 94.5% aa sequence identity (excluding
the signal peptide; supplementary fig. 3A, Supplementary
Material online). In other taxa (e.g., plants, fungi, invertebrates), the p24b1 orthologues are also well conserved
(Carney and Bowen 2004). Moreover, the database queries
revealed that the various members of each of the vertebrate
p24 subfamilies do not only display a high degree of sequence conservation, but also that their genes have similar
intron–exon architectures that correlate well with evolutionary relationships. The genes encoding members of
the related p24a and p24d subfamilies each consist of five
exons, all genes of the related p24b subfamily and the
p24c1/2/5 branch are composed of four exons, whereas
the p24c3/4 genes have only three exons (supplementary
table 2, Supplementary Material online).
FIG. 2.—Tissue distributions of p24 family members. The presence
of transcripts of the p24 family members in mouse tissues was
investigated by RT-PCR. Using two independent primer sets (supplementary table 1, Supplementary Material online), p24a1 was not detected
in any of the tissues examined here. The arrow indicates the position
where the PCR product for p24a1 is expected; the asterisk indicates the
position of the primers used (see also in some of the negative control
lanes). b-Actin served as an input control.
proteins or a limited number of tissues (Denzel et al. 2000;
Kuiper et al. 2000; Rötter et al. 2002; Hosaka et al. 2007).
Therefore, we decided to investigate by RT-PCR the
mRNA expression patterns of all p24 family members in
the mouse. We found that all but two p24 proteins were
widely expressed. Using two different, independent primer
sets, no expression of p24a1 mRNA was found in the 14
tissues examined. For p24c5 mRNA, strong expression
was observed in lung, liver, kidney, small intestine, and colon, and weak expression in spleen, whereas no expression
was found in the other tissues examined (fig. 2). Because no
p24a1 mRNA expression was found in any of the tissues
examined, we searched the database for p24a1 ESTs. Most
p24a1 ESTs were from pancreas (mouse, cow, pig, chicken,
and Xenopus) and a few from liver (dog), placenta (cow), or
embryonic stages (Xenopus), suggesting that the expression
of p24a1 is indeed very limited.
Discussion
Tissue Distributions of the p24 Family Members in the
Mouse
Surprisingly little is known concerning the tissue distributions of the vertebrate p24 proteins. Previous expression studies have examined only a limited number of p24
In this study, we have investigated the composition,
phylogenetic relationship, and expression patterns of the vertebrate p24 family. For most p24 family members, multiple
names are used in parallel, which hampers communication in
the field. Although presently the p23–p27 nomenclature is
1712 Strating et al.
mostly used, it does not offer the flexibility that is needed to
properly describe the full set of p24 proteins that we present
in this paper. Therefore, we suggest employing the systematic nomenclature proposed by Dominguez et al. (1998) and
used in this paper, or at least to provide both names (e.g.,
p23/p24d1). In general, the vertebrate species studied here
contain orthologues of all vertebrate p24 family members.
Our phylogenetic analyses of the complete sets of p24 proteins from man, mouse, zebrafish, Xenopus, fruit fly, yeast,
and Arabidopsis showed that the expansion of the p24 subfamilies has taken place rather recently in evolution, because
it has occurred independently in plants, fungi, and animals,
and partly even independently in vertebrates and invertebrates. Thus, nonvertebrate (plant, fungus, and invertebrate)
p24 proteins are not orthologuous to specific vertebrate family members. Although only a few residues have been conserved throughout the entire p24 family, all p24 proteins
have a similar overall structural organization. Interestingly,
in silico studies predict that the TM domains of the p24a proteins are 3 aa residues shorter than those of the p24b, -c, and
-d subfamily members. The consistency in the positions of
the predicted p24a TM domains suggests that the prediction
is correct. In this connection, it is of interest to note that p24a
proteins localize primarily to the ER, whereas the other
p24s are mainly present in Golgi membranes (Wada et al.
1991; Sohn et al. 1996; Rojo et al. 1997; Dominguez
et al. 1998; Blum et al. 1999; Füllekrug et al. 1999; Gommel
et al. 1999; Emery et al. 2000). Furthermore, the thickness of
secretory pathway membranes increases gradually from the
ER to the plasma membrane, possibly due to an increasing
cholesterol content (Bretscher and Munro 1993). Because
a TM protein may localize correctly by residing in membranes of which the thickness is compatible with the length
of its TM domain (the lipid bilayer thickness model, for review see, Munro 1998), the relatively short TM domains of
the p24a proteins may accommodate their localization in the
ER. However, p24a subfamily members cycle through the
Golgi, and when their retrieval motif is mutated, they
can even reach the late secretory pathway where they form
cholesterol-rich membrane microdomains (Dominguez et al.
1998; Emery et al. 2003). Hence, the TM domain is surely
not the only determinant for the ER localization of p24a proteins. Interestingly, in most p24a subfamily members, the
TM domain is directly followed by two basic and one hydrophobic residue. Possibly, the positive charges can be masked
thus allowing an extension of the p24a TM helix and the presenceofp24a inthickermembranes.Ifandhowthecapabilityof
p25/p24a2 to form cholesterol-rich microdomains relates to
the properties of its TM domain (length, composition) remains
to be established.
We furthermore showed that, with the exception of
p24a1 and p24c5, the mouse p24 proteins are widely expressed, in line with the wide expression of the majority
of p24s in flies (Boltz et al. 2007). We furthermore found
that each tissue examined expressed a representative of
each subfamily and even multiple members of the same
subfamily. The broad coexpression of p24 subfamily members in vertebrate organs has not been noticed before and
may point to a functional nonredundancy, even among
the closest subfamily members. Knowledge of the tissue
distributions of the p24 proteins is also relevant for the de-
sign of functional p24 studies in knockout mice. Because
a homozygous knockout of the widely expressed p23/
p24d1 was lethal in an early embryonic stage (Denzel
et al. 2000), the generation of knockouts of less broadly expressed p24 proteins (such as of p24a1 or p24c5) should be
considered because this approach will increase the chances
that functional p24 studies are feasible. The restricted expression patterns of p24a1 and p24c5 are of special interest
because it may well reflect specialized functions for these
p24 proteins in a restricted number of tissues with ER-toGolgi transport of a specific set of cargo proteins.
Collectively, we present the known vertebrate p24
proteins (all in the systematic nomenclature proposed by
Dominguez et al. 1998), reveal the evolutionary conservation of the entire p24 family in line with previous reports on
smaller subsets of the p24 family (Stamnes et al. 1995;
Fiedler and Rothman 1997; Dominguez et al. 1998; Emery
et al. 1999; Marzioch et al. 1999; Kuiper and Martens 2000;
Kuiper et al. 2000; Rötter et al. 2002; Carney and Bowen
2004), show the tissue distributions of all 10 mouse p24
family members, and report the discovery of a novel family
member that displays a restricted expression pattern. Together, we provide for the first time a comprehensive overview of the vertebrate p24 family. As such, our findings will
help in the design and interpretation of future studies aimed
at elucidating p24 functioning.
Supplementary Material
Supplementary figures 1–3 and supplementary tables
1 and 2 are available at Molecular Biology and Evolution
online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
This work was supported by grant 811.38.002 from the
Netherlands Organisation for Scientific Research—Earth
and Life Sciences (NWO-ALW). We would like to thank
the members of the Martens laboratory for helpful discussions and encouragements, and Frank Oerlemans, Eric
Jansen, and Jessica van Schijndel for help with the dissection
of mouse tissues.
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Accepted April 9, 2009