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A New Subfamily of Major Intrinsic Proteins in Plants
Urban Johanson and Sofia Gustavsson
Department of Plant Biochemistry, Lund University, Sweden
The major intrinsic proteins (MIPs) form a large protein family of ancient origin and are found in bacteria, fungi,
animals, and plants. MIPs act as channels in membranes to facilitate passive transport across the membrane. Some
MIPs allow small polar molecules like glycerol or urea to pass through the membrane. However, the majority of
MIPs are thought to be aquaporins (AQPs), i.e., they are specific for water transport. Plant MIPs can be subdivided
into the plasma membrane intrinsic protein, tonoplast intrinsic protein, and NOD26-like intrinsic protein subfamilies.
By database mining and phylogenetic analyses, we have identified a new subfamily in plants, the Small basic
Intrinsic Proteins (SIPs). Comparisons of sequences from the new subfamily with conserved amino acid residues
in other MIPs reveal characteristic features of SIPs. Possible functional consequences of these features are discussed
in relation to the recently solved structures of AQP1 and GlpF. We suggest that substitutions at conserved and
structurally important positions imply a different substrate specificity for the new subfamily.
Introduction
Major intrinsic proteins (MIPs) are integral membrane proteins that facilitate the passive transport of
small polar molecules across membranes. In many membranes, MIPs are very abundant and constitute the major
membrane protein. Plant MIPs have been classified into
three different subfamilies in phylogenetic comparisons
(Weig, Deswartes, and Chrispeels 1997; Johansson et al.
2000). Two of the subfamilies, plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins
(TIPs), are named according to the subcellular location
of the proteins. The third group of MIPs shows similarity with NOD26, a nodulin expressed in peribacteroid
membrane surrounding the symbiotic nitrogen-fixing
bacteria in nodules of soybean roots (Fortin, Morrison,
and Verma 1987). These proteins are called NOD26-like
MIPs (NLMs; Weig, Deswartes, and Chrispeels 1997)
or NOD26-like intrinsic proteins (NIPs; Heymann and
Engel 1999).
Most of the plant MIPs that have been tested have
been shown to function as water channels (for recent
reviews see Johansson et al. 2000 and Santoni et al.
2000). However, there are some examples of plant MIPs
that show a broader permeability. For example Nt-AQP1
(aquaporins [AQP]) belongs to the PIPs and transports
water, glycerol, and urea, whereas Nt-TIPa mainly transports urea but also water and glycerol. Several NIPs
have been shown to transport both water and glycerol
(Dean et al. 1999; Weig and Jakob 2000). The recent
determination of the structure of the water-specific AQP1
and the glycerol-specific GlpF to a resolution of 3.8 and
2.2 Å, respectively, has identified residues that are important determinants for the substrate specificity (Fu et
al. 2000; Murata et al. 2000). These structures provide
valuable information for predicting the substrate specificity of new forms of MIPs.
The genome sequencing project of Arabidopsis has
revealed many new genes (Arabidopsis Genome InitiaKey words: MIPs, water channels, aquaporins, glycerol facilitators, SIPs.
Address for correspondence and reprints: Urban Johanson, Department of Plant Biochemistry, P.O. Box 124, S-221 00 Lund, Sweden. E-mail: [email protected].
Mol. Biol. Evol. 19(4):456–461. 2002
q 2002 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
456
tive 2000). The new genes are automatically annotated
according to their best matches in similarity searches,
and in most cases this annotation will provide a clue to
their function. However, sometimes, for example, when
the distance to a well-characterized protein is too long,
this fast annotation fails or is directly misleading. For
example, accession AAC26712 has been annotated as a
PIP, although it clearly belongs to the recently discovered NIP subfamily (NIP2;1, Johanson et al. 2001). To
facilitate a correct annotation of genomic or EST accessions, to a reasonable resolution, it is important to perform phylogenetic analyses of protein families to identify the major branches in the phylogenetic tree. The aim
of this paper is to present characteristic features of the
Small basic Intrinsic Proteins (SIPs), a new and novel
plant MIP subfamily.
Materials and Methods
SIPs genes and ESTs were found by BLAST or
TBLASTN searches at the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov:80/
blast/blast.cgi) or at The Arabidopsis Information Resource (TAIR; www.arabidopsis.org/blast/).
MacVector 7.0 (Oxford Molecular Ltd, U.K.) was
used to translate sequences and to calculate pIs and molecular weights presented in figure 2. ClustalW (Thompson, Higgins, and Gibson 1994), included in MacVector
7.0, was used to generate multiple alignments of translated sequences using the blosum matrix and slow mode.
Open gap penalty and extend gap penalty were set to
10.0 and 0.05, respectively. Alignments were manually
inspected and adjusted to fit to conserved residues and
to avoid gaps in transmembrane helices (Heymann and
Engel 2000). The alignment of SIPs to GlpF and AQP1
is unambiguous in all helices except in helix 2 and 5.
These regions are harder to align because of the lack of
conserved residues that are shared between SIPs and
other MIPs.
PAUP*4.0b4a (Swofford 2000) was used in phylogenetic analyses. Cytoplasmic N- and C-termini were
excluded from the phylogenetic analyses of the 374character–long alignment. Cytoplasmic N- and C-termini were defined as characters 1–92 and 336–374, respectively, based on the alignment with AQP1 and GlpF.
SIPs a New Subfamily of MIPs
457
FIG. 1.—Phylogenetic comparison of protein sequences of the new SIP subfamily of MIPs to representatives for the previously described
subfamilies of Arabidopsis PIPs, TIPs, and NIPs. This unrooted phylogram has been generated in PAUP4.0b using the distance method with
minimum evolution as the optimality criteria. Parsimonious analysis results in one tree (data not shown) with a very similar topology to the
shown distance tree. The only difference when compared with the distance tree is the position of TIP3;1 and TIP3;2 which branch closer to
TIP1s in the parsimony tree. Each of the four subfamilies are monophyletic, regardless of method, with bootstrapping values of 98%–100%
depending on the tree building method used. Protein accession numbers are found in Johanson et al. (2001). Cri BE641624, Gar AW729182,
and Hvu AW982441 denote proteins translated from ESTs (table 1). AW982441 probably encodes the full-length protein, whereas the other two
ESTs probably are slightly truncated in the C termini (no stop codon in the ESTs).
One hundred bootstrap replicates were performed in
bootstrap tests using the option full heuristic search.
RasMac Molecular Graphics 2.6 (Sayle 1995) was
used to inspect the structure of GlpF and AQP1, PDB
code 1FX8 and 1FQY, respectively, and also to generate
figure 4.
Results and Discussion
Phylogenetic Analysis and ESTs
Blast searches in the Arabidopsis genomic sequence (Arabidopsis Genome Initiative 2000) reveal
three novel MIP-like genes, T6K12.29, MRG7.25, and
F24I3.30, all very different from any of the previously
described plant MIP genes belonging to the PIP, TIP, and
NIP subfamilies (U. Johanson at the MIP 2000 meeting
in Göteborg, Sweden, unpublished data). Phylogenetic
comparison with Arabidopsis representatives for these
three MIP subfamilies shows that the new proteins form
a distinct and new subfamily of MIPs (fig. 1). T6K12.29
and MRG7.25 are more similar to each other than to the
F24I3.30. We suggest that these proteins should be
named SIP1;1, SIP1;2, and SIP2;1, respectively (see
subsequently and Johanson et al. 2001). Matching ESTs
in Arabidopsis verify that all three new genes are expressed and therefore likely to be fully functional (table
1). Judging from the low number of corresponding ESTs
(2–6/gene), the overall expression levels of the new
genes are likely to be much lower than for most PIPs
and TIPs which have very abundant mRNAs (Weig,
Deswartes, and Chrispeels 1997).
ESTs encoding SIP-like proteins can not only be
identified in Arabidopsis and other dicotyledons but also
in several monocotyledons, one conifer, and one fern
species (Ceratopteris richardii). This suggests that SIPs
are widely distributed in the plant kingdom and can be
found at least in all higher vascular plants. On the basis
of the partial sequence of the ESTs, most of them can
be classified as SIP1s; only one (Glycine max
AW351321) is definitely of the SIP2 type (data not
shown). According to the ESTs, SIP2s are expressed
preferentially in roots, whereas there is no obvious pattern in the expression of SIP1s. Very recently, a phylogenetic analysis of different MIPs identified in
470,000 maize ESTs revealed that maize also has at least
one SIP2 gene in addition to two SIP1 genes (Chaumont
et al. 2001).
Characteristics of SIPs
We have previously noted that the different MIP
subfamilies tend to have characteristic biochemical
properties (Johansson et al. 2000). For instance, TIPs
are in general smaller and more acidic than PIPs or
NIPs. Proteins of the new subfamily are also small like
TIPs but still different from TIPs in that they are highly
basic proteins (fig. 2). Hence, we suggest the name SIPs
for this new MIP subfamily. The main reason for their
458
Johanson and Gustavsson
Table 1
SIP-like EST from Plants and Their Minimal Expression Pattern Based on Information in the Sequence Accessions
Species
Ceratopteris richardii . . . . . . .
Pinus taeda. . . . . . . . . . . . . . . .
Zea mays. . . . . . . . . . . . . . . . . .
Oryza sativa . . . . . . . . . . . . . . .
Hordeum vulgare . . . . . . . . . . .
Secale cereale . . . . . . . . . . . . .
Medicago truncatula . . . . . . . .
Gossypium arboreum. . . . . . . .
Glycine max . . . . . . . . . . . . . . .
Lycopersicon esculentum. . . . .
Solanum tuberosum . . . . . . . . .
Arabidopsis thaliana . . . . . . . .
Accession
Number
BE641624
AW010799
AI668312
AI667776
C99574
AW982441
BE494650
AW696882
AW574071
AW729182
AI440840
AW350302
AW508796
AW351321
AI485307
AI484774
AI780558
AW625404
AW039249
BE341068
AI997475
AV523893
AV550067
AV525526
AV537365
AA404750
AV538923
AV549528
AV541085
AV543784
Typea
2
1;1
1;1
1;2
1;2
2;1
2;1
2;1
2;1
2;1
2;1
Tissue
Time/Stageb
Germinating spores
Shoot tips
Endosperm
Endosperm
Panicle
Spike
Anther
Stem
Nitrogen-fixing root nodules
Balls, fiber
Root
Root
Cotyledons
Root
Carpel
Carpel
Leaf
Radicle
Leaf 1 elicitors
Axillary buds of stem
Root
Above ground
Root
Above ground
Root
Mixture include root
Root
Root
Root
Root
20 h
Spring
10–14 d pollination
10–14 d pollination
.10 cm
Preanthesis
Preanthesis
Mixture
1 month after inoccul.
8d
8d
Immature
8d
5 d pre- to 5 d
Postanthesis
4 week old
5 d postimbibition
4–6 week old
1–3 d
4–7 week old
2–6 week old
2–6 week old
a Type compared to the Arabidopsis thaliana SIPs 1;1, 1;2, and 2;1. Most of the ESTs from species other than Arabidopsis thaliana are SIP1-like (not indicated),
only one is definitely of SIP2-type.
b Abbrevations: d, days; h, hours.
small size is a very short cytosolic N-terminal region
compared with the other plant MIPs. The N-terminal
region in SIPs is even shorter than in TIPs but similar
to AqpZ from Escherichia coli. The high isoelectric
point of SIPs is partly caused by runs of lysines in the
C-terminal region. Unlike in the basic PIPs and NIPs,
these basic residues are not a part of any evident phosphorylation site (Johansson et al. 2000).
FIG. 2.—Comparison of molecular weight and isoelectric point
for representatives of different subfamilies of MIPs from Arabidopsis.
For accession numbers, see figure 1.
Heymann and Engel (2000) have compiled and
aligned in total 164 different forms of MIPs from bacteria, fungi, animals, and plants. On the basis of 46 different type sequences they identified highly conserved
amino acid residues. The high degree of conservation
suggests that all MIPs have a common fold, and that the
conserved residues have a crucial role in the structure
and function of most MIPs. The recently published
structures of the human water channel AQP1 and the
bacterial glycerol facilitator GlpF are indeed very similar, despite the difference in substrate specificity and the
long evolutionary distance between man and bacteria
(Fu et al. 2000; Murata et al. 2000). The SIPs clearly
belong to the MIP family because they have most of the
conserved amino acid residues found in other MIPs.
However, there are several interesting deviations in SIPs
compared with other MIPs that are worth commenting
on and on which this paper will focus.
The predicted topology of SIPs conform to the general structure of MIPs with six transmembrane helices
and two shorter transmembrane helices that together
form a seventh transmembrane region connected by two
NPA or NPA-like motifs (fig. 3). Thus, the overall fold
of the SIPs is likely to be similar to other MIPs like
AQP1 or GlpF. Both AQP1 and GlpF form right-handed
helical bundles around the pore with a narrow part close
to the asparagines of the NPA boxes. In GlpF, the in-
SIPs a New Subfamily of MIPs
Table 2
Structurally Important Amino Acids in MIPs and Their
Role in the Structure of GlpF or AQP1 (or both)
Residue GlpF
(AQP1)a
SIPsb
E14. . . . . . . . .
S63 . . . . . . . . .
H66 . . . . . . . .
F89 (Y) . . . . .
A70 . . . . . . . .
V71 . . . . . . . .
T72. . . . . . . . .
D207 (S) . . . .
P210 (S). . . . .
R206. . . . . . . .
L21 (F) . . . . .
I187 (V). . . . .
W48 (F) . . . . .
F200 (C) . . . .
G49 . . . . . . . .
G184 . . . . . . .
P180 . . . . . . . .
D
G
S
R
T
G
S
A
W
I/N
W
T
L/S
P
L
S
T
Role in GlpF/AQP1c
Stabilizing loop B
Stabilizing loop B
Stabilizing loop B
Stabilizing loop B
Packing core near N68
Packing core near N68
Packing core near N68
Packing core near N203
Packing core near N203
Hydrogen bond donor, selective filter G2
Hydrophobic lining
Hydrophobic lining
Hydrophobic corner
Hydrophobic corner
Packing helix 2 and 5
Packing helix 2 and 5
Packing helix 2 and 5
a Amino acid and position in GlpF. Corresponding amino acid in AQP1 is
given brackets when it is different.
b Corresponding consensus amino acid residues in SIPs, from figure 3, are
shown for comparisons. When corresponding amino acid residue is not conserved, the two dominating amino acid residues are given.
c Structural role in GlpF-AQP1 (Murata et al. 2000; Fu et al. 2000).
459
terior of the channel is mainly hydrophobic, with the
exception of a helical polar stripe running through the
channel providing sites of hydrophilic interactions for
the hydroxyl groups of glycerol. The polar stripe consists of carbonyls from the backbone of the extended
chain preceding the two short half transmembrane helices, the side chains of asparagine of the two NPA boxes, and a conserved arginine (206) in the second half
transmembrane helix (Fu et al. 2000).
Some structurally important positions where SIPs
are different compared to GlpF and AQP1 are listed in
table 2, and in figure 4 some of these positions are
shown in the structure of GlpF. In general, the amino
acid residues at these positions are conserved among
non–SIP-MIPs. Several deviations from the MIP consensus are clustered in positions that affect loop B and
helix B. In helix 1, there is a very conserved glutamate
(E14) among MIPs. The corresponding amino acid in
all SIPs investigated is aspartate. In GlpF, the carboxyl
group of E14 is important in fixating loop B in the right
position by forming a hydrogen bond to the backbone
NH of H66 (Fu et al. 2000). If the same interaction were
maintained in SIPs, this would change the position of
loop B relative to helix 1 and might result in a wider
cytoplasmic vestibule. Three residues before H66 in
GlpF there is a conserved S63. The corresponding S71
in AQP1 is thought to further stabilize loop B by hydrogen bonding to Y97 in helix 3. This interaction is
FIG. 3.—Alignment of GlpF and AQP1 to the SIPs included in the phylogenetic analysis. Dark and light shadings show positions with
identical and similar amino acid residues, respectively, shared by at least six of the eight aligned sequences. Transmembrane helices common
to both AQP1 and GlpF are boxed and numbered H1–H6. HB and HE together form a seventh transmembrane helix, connected by the two
NPA boxes. The most common amino acid residue among MIPs, according to figure 3 in Heymann and Engel (2000) is shown at the top for
some positions where SIPs differ from other MIPs. At the bottom, the SIP consensus at these positions is indicated. P4–P5 mark two positions
that have been suggested to be important determinants of substrate specificity for water and glycerol channels (Froger et al. 1998).
460
Johanson and Gustavsson
FIG. 4.—Part of the interior structure of GlpF, showing the asparagines 68 and 203 in the NPA boxes and some of the discussed amino
acid residues as ball-and-stick models (Fu et al. 2000). Oxygen, nitrogen, and carbon atoms are depicted in dark, intermediate, and light
gray, respectively (in the on-line version, oxygen and nitrogen atoms
are visualized in red and blue, respectively). The top of the structure
is facing periplasmic space and the arrow indicates the orientation of
the pore. The side chains of N68, N203, and R206, together with the
carbonyl oxygen of H66 form part of the helical polar strip that interacts with hydroxyl groups of glycerol. I187 and F200 form part of the
hydrophobic back of the pore and the hydrophobic corner, respectively,
that interacts with the alkyl backbone of glycerol. In SIPs, I187 is
replaced by a threonine, changing the pattern of polar residues in the
pore. The structure of the hydrophobic pocket is changed by the replacement of F200 for a proline in most SIPs. R206 is conserved in
almost all MIPs. However, the corresponding amino acid in SIPs is not
conserved but varies between hydrophobic residues like isoleucine to
polar residues such as asparagine. The carbonyl oxygen of H66 constitutes another polar interaction site in the pore of GlpF. The position
of H66 is stabilized by E14. This glutamate is replaced by the one
carbon shorter aspartate in all SIPs, possibly changing the position of
the serine in SIPs corresponding to H66. This might result in a wider
pore. The changed interior environment suggests that SIPs have a different substrate specificity compared with other MIPs.
not possible in SIPs where the corresponding residues
are G and R, respectively, although other types of interactions cannot be excluded. Positions A70–T72 are
substituted in most of the SIPs examined. These positions are part of the core close to N68 in the first NPA
box and may be of importance for the correct positioning of N68. D207 and P210 probably have a corresponding function in helix E, in that they influence the position of N203 in the second NPA box. The highly conserved R206, also in helix E, is not present in SIPs. In
GlpF, the arginine side chain is part of a glycerol-binding site which has been proposed to constitute a part of
a selectivity filter responsible for the glycerol specificity
(Fu et al. 2000). Interestingly, a V or S mutation of the
corresponding R189 in the bacterial water channel AqpZ
abolishes water transport (Borgnia et al. 1999). F200 is
not conserved among MIPs and is replaced by P in most
SIPs. All these changes may affect the specificity of the
channel because positions of the carbonyls of H66 and
F200, together with the side chains of N68, N203, and
R206, determine the geometry of part of the polar strip
inside the channel. Furthermore, some of the hydrophobic residues inside the GlpF channel are changed in SIPs
and may in some cases form hydrogen bonds to a transported molecule. In particular, the threonine in SIPs at
the position corresponding to I187 in GlpF is interesting
because this residue might disrupt the hydrophobic back
and provide a novel polar patch directly opposite to N68
and N203, again suggesting that SIPs have a different
specificity compared with other MIPs.
In GlpF, the aromatic W48 and F200 form a hydrophobic corner that interacts with the alkyl chain of
glycerol (Fu et al. 2000). In SIPs, they are replaced by
the smaller nonaromatic L or S and P, respectively. The
most conserved amino acid residues in helices 2 and 5
are G49 and G184, respectively. These residues allow a
close packing of these two helices. In SIPs, the G residues are substituted by L and S, respectively, which will
affect the packing of helices 2 and 5. The result of these
changes could be a wider pore. In addition, the lessconserved P180 in helix 5 is also involved in the interaction with helix 2 because it protrudes from helix 5
and fits nicely in between I56 and Y57 of helix 2. It is
hard to predict the effects of the polar threonine at this
position in the SIPs. It might affect the packing of helix
2 and 5, but it is also possible that the hydroxyl group
is pointing to the cytoplasmic vestibule, thereby providing another hydrophilic interaction site for the substrate.
However, it is important to realize that the exact alignment, of SIPs to GlpF and AQP1, in helix 2 and 5 is
not as clear as in other regions because of the lack of
the conserved glycines in SIPs.
In summary, the SIPs are different at many important positions that are likely to affect the interior properties of the channel. Both the width of the pore and the
hydrophilic-hydrophobic pattern inside the channel may
be altered in SIPs, as compared with other MIPs. This
suggests that SIPs have a different substrate specificity
than both AQP1 and GlpF. From sequence comparisons,
it has previously been suggested that there are mainly
five positions, P1–P5, that are important determinants of
substrate specificity for water and glycerol channels
(Froger et al. 1998). Remarkably, mutating a YW to PL
at P4–P5 has been shown to be sufficient to convert one
water channel, AQPcic, into a glycerol facilitator (Lagrée et al. 1999). In this context, it is interesting to note
that all the SIPs carry the canonical aquaporin sequence,
YW, at the corresponding position.
Function and Intracellular Location of SIPs
We have identified a new type of MIP that is very
different from other previously identified plant MIPs. In
order to elucidate the function of SIPs, it is important
to determine their intracellular localization, expression
pattern, and substrate specificity. Experiments are in
progress to address these questions. In the meantime, it
is important to recognize the SIPs as a new subfamily
of plant MIPs and to avoid confusing them with other
SIPs a New Subfamily of MIPs
MIPs because the SIPs most likely have a different function compared with all previously characterized MIPs.
Acknowledgments
This work was funded by NFR, SJFR and EU-Biotech program (BIO4-CT98-0024). We are grateful to Per
Kjellbom and Salam Al-Karadaghi for critical reading
and helpful suggestions on the manuscript.
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Accepted November 20, 2001