(Received for publication, October 2, 1996, and in revised form, April 15, 1997)
From the Departments of Genetics and Cell Biology and Botany, Washington State University, Pullman, Washington 99164-4238
Photoaffinity labeling of a soybean cotyledon membrane fraction identified a sucrose-binding protein (SBP). Subsequent studies have shown that the SBP is a unique plasma membrane protein that mediates the linear uptake of sucrose in the presence of up to 30 mM external sucrose when ectopically expressed in yeast. Analysis of the SBP-deduced amino acid sequence indicates it lacks sequence similarity with other known transport proteins. Data presented here, however, indicate that the SBP shares significant sequence and structural homology with the vicilin-like seed storage proteins that organize into homotrimers. These similarities include a repeated sequence that forms the basis of the reiterated domain structure characteristic of the vicilin-like protein family. In addition, analytical ultracentrifugation and nonreducing SDS-polyacrylamide gel electrophoresis demonstrate that the SBP appears to be organized into oligomeric complexes with a Mr indicative of the existence of SBP homotrimers and homodimers. The structural similarity shared by the SBP and vicilin-like proteins provides a novel framework to explore the mechanistic basis of SBP-mediated sucrose uptake. Expression of the maize Glb protein (a vicilin-like protein closely related to the SBP) in yeast demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake. Thus, despite sequence and structural similarities shared by the SBP and the vicilin-like protein family, the SBP is functionally divergent from other members of this group.
The development of plant seeds involves the accumulation of carbon
and nitrogen reserves in proteinaceous forms that can both withstand
desiccation and be utilized as an energy source by the developing
embryo during germination. In legume species, these predominant seed
storage proteins are found enclosed in membrane-bound organelles known
as protein bodies (1, 2). The globulin seed storage proteins generally
fall into two main classes: legumin-like and vicilin-like proteins (1,
3-6). Under nonreducing conditions, the legumin-like proteins are
found as hexameric complexes with sedimentation coefficients of 11 S. The subunits of these complexes are derived from a precursor peptide
containing two domains: an N-terminal acidic chain and a C-terminal
basic
chain. Following proteolytic processing, these domains remain
associated through interchain disulfide links. In contrast, the
vicilin-like proteins are found as 7 S trimers under nonreducing
conditions. The vicilin-like monomers are 50-70-kDa polypeptides that
undergo variable levels of post-translational proteolytic
processing (1, 3).
X-ray crystallography of the vicilin-like proteins phaseolin (7, 8) and
canavalin (9, 10) has permitted the formation of a canonical
three-dimensional model for vicilin-like molecules (8). Each monomer
consists of two very similar structural domains reflecting a tandem
duplication observed at the nucleotide and amino acid sequences. These
tandem domains are composed of two structural elements: a compact
eight-stranded barrel having the "Swiss roll" topology and an
extended loop containing several short
helices. Trimers are formed
by the apposition of a hydrophobic surface composed of side chains from
one face of the N-terminal domain and a complementary hydrophobic
surface of the C-terminal domain of the neighboring monomer. These
intermonomeric interactions are further stabilized by several salt
bridges. Once formed, the trimer is a disc-shaped molecule with a large
internal channel of 18 Å (8, 10). The high degree of structural
conservation shared by these two vicilin-like proteins is likely
the result of several evolutionary constraints including subunit
interactions that are required for holoprotein assembly, packaging of
holoproteins into protein bodies, tolerance to desiccation, sorting in
the secretory pathway, and general constraints for stability and
solubility (11-14).
The accumulation of carbon in developing seeds is mediated by specific plasma membrane proteins (15-17). In most legume species, sucrose is the predominant form of assimilated carbon translocated from the photosynthetically active leaves to the developing cotyledons (18, 19). Photoaffinity labeling of membranes isolated from soybean cotyledon tissue with a photolyzable sucrose analog identified a distinct 62-kDa sucrose-binding protein (SBP)1 (20). Analysis of the cDNA encoding the SBP and its deduced amino acid sequence indicates that the SBP contains a single hydrophobic domain at its N terminus but otherwise is a hydrophilic protein lacking the expected membrane-spanning hydrophobic segments typically present in transport proteins (21). Biochemical analysis of the topology of the SBP demonstrates that it is tightly associated with the external leaflet of the plasma membrane (22). The involvement of the SBP in sucrose uptake was implicated by immunolocalization experiments, which demonstrated that the SBP is exclusively associated with the plasma membrane of cells involved in active sucrose uptake (21). Kinetic analysis of SBP-mediated sucrose uptake in a yeast system indicates that the uptake is specific for sucrose but is proton-independent and relatively nonsaturable, thus defining a novel mechanism for sucrose uptake (15).
Despite strong evidence that the SBP mediates sucrose uptake, nucleotide and amino acid sequence analysis suggests that the SBP is distinct from other known transport proteins including binding protein-dependent transport systems of Escherichia coli. Here we show that a portion of the SBP amino acid sequence shares significant sequence homology with the vicilin-like seed storage proteins, including an internal sequence repeat. In addition, examination of plasma membrane fractions under nonreducing conditions show that the SBP exists in multimeric complexes that may represent dimeric and trimeric association of SBP monomers. Thus, the data presented here provide new insight into the three-dimensional organization of a unique plasma membrane protein capable of mediating sucrose uptake.
Soybean (Glycine max common version Wye) seeds were grown as described (21). Microsomal membranes from cotyledons were obtained at the indicated times after flowering or imbibition following the protocol of Overvoorde and Grimes (22). Highly enriched plasma membrane proteins were obtained by aqueous two-phase partitioning as described previously (22-25).
RNA Isolation and Gel Blot AnalysisCotyledons were removed
from the pod walls or parts of the germinating embryo, frozen in liquid
nitrogen, and stored at 80 °C. RNA was isolated using the protocol
described by Grimes et al. (21). For analysis of
sbp mRNA levels, the probe was a 1.7-kilobase pair
NcoI/SpeI fragment of the sbp cDNA
(21) labeled with 32P by random priming with a RadPrime
(Life Technologies, Inc.) kit according to the manufacturer's
directions. For analysis of
-conglycinin mRNA levels, a 350-base
pair polymerase chain reaction fragment amplified from soybean genomic
DNA was labeled with 32P by random priming. Direct
sequencing of this polymerase chain reaction fragment on an ABI 373 sequencer (Applied Biosystems, Inc.) confirmed the identity of this
probe. Membranes containing the transferred RNA were prehybridized at
65 °C for 1 h in 1% SDS, 1 M sodium chloride, and
10% dextran sulfate. Hybridizations were carried out overnight at
65 °C in the same buffer containing 1-5 ng/ml denatured probe and
100 µg/ml denatured salmon sperm DNA. The blots were washed twice
with 2 × SSC (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) for 5 min each with constant agitation at room temperature, twice with 2 × SSC, 1% SDS for 30 min each at 65 °C, and twice with 0.1 × SSC for 15 min each at
room temperature.
Purified plasma membrane
proteins were solubilized in Buffer A (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA) supplemented with 1%
Triton X-100 for 30 min on ice. This suspension was clarified by
centrifugation at 100,000 × g
(ravg) for 24 min at 4 °C in a Beckman TL-100
ultracentrifuge. >85% of the membrane proteins were solubilized by
this treatment. The supernatant was loaded onto a 5-25% sucrose
gradient containing Buffer A supplemented with 0.1% Triton X-100.
After centrifugation at 4 °C for 16 h at 130,000 × g (ravg) in a Beckman SW40 rotor,
fractions of 0.5 ml were collected from the bottom. One-fifth of each
fraction was diluted with an equal volume of buffer containing 50 mM Tris, pH 6.8, 2% SDS, and 1 mM
-mercaptoethanol, collected on nitrocellulose using a slot-blot
apparatus (Hoefer Scientific Instruments), and immunostained using
affinity-purified antiserum described by Grimes et al. (21).
Densitometry was performed as described earlier (22).
Data base searches and sequence alignments were carried out using the GCG software package (26). Unrooted phylogenetic trees were generated using the neighbor-joining and fitch algorithms contained in the phylogenetic inference package, PHYLIP 3.57 (27).
Expression of SBP Forms and Glb1 in Yeast and Analysis of [14C]Sucrose UptakeTo facilitate the expression of Glb1 in yeast, pYEGlb1 was constructed by excising the glb1 cDNA from pGlb1 (28, 29) by digestion with SalI and BamHI and ligation of this fragment into the XhoI and BamHI sites of pMK195 (15). The construct was confirmed by restriction digests. Two forms of the SBP were tested as well. A SBP with its C terminus truncated was excised from pSBP using XhoI and XbaI sites, and this fragment was ligated into the XbaI and XhoI sites of pMK195. This construct resulted in the deletion of 80 amino acids from the C terminus. A full-length SBP was excised from pSBP using SalI and PstI and ligated into the PstI and XhoI sites of pMK195. The truncated form was used in a previous study (15).
susy7/ura3 yeast (15, 16) were transformed with 1 µg of pMK195, truncated pYESBP (15), full pYESBP, or pYEGlb1 using a small scale, lithium acetate-based protocol (30). To characterize the putative transformants, DNA was isolated (32) and analyzed either by Southern blot analysis (31) or by being electroporated into XL-I Blue E. coli. Plasmid DNA from transformed E. coli colonies growing on solidified Luria broth medium containing 100 µg/ml ampicillin was isolated (31) and used to confirm the structure of the re-isolated shuttle vectors by restriction digests. The yeast strain termed OG2 contains the C-terminal truncated SBP, whereas CG1 contains the full-length SBP. Both of these constructs were verified by DNA sequencing.
Colonies containing the appropriate vectors were grown to an A600 of 0.5-1.0 in liquid minimal medium supplemented with His, Trp (ura dropout). After harvesting by centrifugation, the cells were washed twice with 25 mM Mes-KOH buffer, pH 5.5, weighed, and resuspended in the same buffer to a density of 30-50 mg of cells/ml. [14C]Sucrose uptake assays were performed as described by Overvoorde et al. (15).
Analytical MethodsProtein concentration was assayed with
the bicinchoninic acid reagent according to the manufacturer's
directions (Pierce). Polyacrylamide gels were run according to Laemmli
(33), except that a 7.5-15% acrylamide gradient was used with an
accompanying 7.5-15% glycerol gradient. To determine the mobility of
the SBP under nonreducing conditions, the samples were solubilized in sample buffer lacking -mercaptoethanol but containing SDS and were
not heated before loading. Gels were stained with 0.2% Coomassie Brilliant Blue R-250. For immunostaining, proteins resolved by SDS-PAGE
were electroblotted to nitrocellulose (34) and immunodecorated using
affinity-purified anti-SBP antisera as described by Grimes et
al. (21). Color development was performed using ECL according to
the manufacturer's directions (Amersham Corp.).
Analysis of the
deduced amino acid sequence of the SBP shows that it lacks similarity
with any other known transport protein identified from other species
(21). Searches of the GenBankTM data base for SBP
homologues, however, reveal that the SBP shares 44-61% similarity and
20-37% identity with the vicilin-like seed storage proteins
(Fig. 1). The vicilin-like family of proteins shares
several conserved features, and a detailed comparison of the SBP
sequence with other members of this family highlights the close
relationship of the SBP with this group of polypeptides.
Seed storage proteins typically contain a hydrophobic signal sequence that targets the newly synthesized polypeptides to the endoplasmic reticulum where the protein undergoes a variety of modifications, including the proteolytic removal of the signal sequence, glycosylation, and sorting for transit through the remainder of the secretory pathway. Similarly, the SBP contains a hydrophobic stretch of amino acids at its N terminus that may function as a signal sequence. Microsequencing of the N terminus of the mature SBP (21), however, shows that if this hydrophobic domain is removed, the site of processing (indicated by an asterisk in Fig. 1) is different from the highly conserved site of processing shared by both vicilin- and legumin-like proteins (indicated by a down arrow in Fig. 1; Ref. 4).
The vicilin-like proteins can be roughly grouped into two size classes depending on the length of a variable highly charged segment that follows the hydrophobic N terminus (35). Storage proteins containing this hydrophilic segment (which ranges in size from 90 to 183 residues) are approximately 70 kDa, whereas those lacking this region are approximately 50 kDa. Although considerably smaller (73 residues) than the 70-kDa proteins, the SBP contains a similar hydrophilic segment that is rich in charged amino acids, especially Glu and Arg (Fig. 1; Ref. 21). In addition, the CXXXC motif that is repeated six times in this portion of the cotton vicilin and twice in the vicilin-like storage proteins of cocoa, barley, soybean, wheat, and maize is also present twice in the SBP sequence (residues 43-47 and 61-65 using SBP numbering in Fig. 1; Refs. 28 and 35-39). Thus, the SBP shares both the charged hydrophilic domain and the conserved CXXXC motif within this domain with other members of the vicilin-like proteins.
A third shared feature of the SBP with the vicilin-like proteins is the presence of a repeated sequence that is thought to have arisen through the duplication of a primordial gene (5, 6, 8, 40). The three-dimensional structure of both phaseolin and canavalin, vicilin-like proteins from Phaseolus vulgaris and Canavalia ensiformis, respectively, demonstrates that this duplicated sequence forms the basis for the symmetrically reiterated Swiss roll domain structure (7-10). Alignment of the SBP sequence with phaseolin and canavalin shows that the SBP contains a similar internal repeat (residues 117-299 and 310-509; Fig. 1).
Lawrence et al. (8) identified 26 strictly conserved
residues present in all members of the vicilin-like protein family (indicated by a black circle under the sequences in Fig. 1).
Of these 26 residues, 23 (88%) are also present in the SBP sequence (Fig. 1). Analysis of these conserved residues in the context of the
known three-dimensional structures demonstrates their importance in
maintaining the overall structure and intramonomer contacts (8). As
such, the SBP appears likely to contain similar tertiary motifs and to
be organized in similar three-dimensional structures. Two potentially
significant departures from this conservation are the substitution of
Ile for Pro at position 361 of the SBP (36) and the substitution of Ala
for Pro at position 157 of the SBP. Both of these Pro residues result
in a bulge forming in the respective
sheets, and these
bulges are postulated to be important in mediating oligomeric formation
(10).
Within the vicilin-like sequence, there are segments that highlight the relatedness of the reiterated motif (10). For instance, residues 173-181 (canavalin numbering) in the N-terminal domain of canavalin share 67% similarity (44% identity) with its C-terminal counterpart, residues 375-383 (10). Such similarity and that of adjoining regions support the hypothesis that these domains have arisen by tandem duplication of a smaller ancestral protein (5-10). Interestingly, the SBP contains residues that are conserved between its two domains that are not conserved in other vicilin-like sequences (e.g. Phe-239 and Gly-243 (SBP numbering) in the N-terminal domain are found in the same register at positions 449 and 453, respectively, as in the C-terminal domain). The significance of these observations for inferring the evolutionary relationship of the SBP and other proteins that contain vestiges of this repeated domain remains unclear.
An additional conserved feature of the vicilin-like proteins is the LQRF motif at positions 70-73 of the canavalin sequence and positions 60-63 of the phaseolin sequence. This LQ(R/K)F motif is similar to the yeast carboxypeptidase Y vacuolar targeting signal and has been proposed to function as a vacuolar targeting motif in the vicilin-like proteins as well (9). This region in the SBP is LKKF, which may partially explain the targeting of the SBP to the plasma membrane rather than vacuoles or protein storage bodies.
SBP and-Conglycinins are vicilin-like storage proteins
found in soybean cotyledons (14, 41). The expression, processing,
localization, and hydrolysis during germination of these storage
proteins are well defined (14, 42). A direct comparison of
-conglycinin and SBP mRNA levels shows that their expression
patterns are regulated in a similar manner during seed development.
Both transcripts are present in cotyledons 10 days after flowering and
reach a peak at 18-22 days after flowering (Fig. 2).
After this time, both sbp and
-conglycinin mRNA
levels decrease to undetectable levels at 26-28 days after flowering.
During germination, neither of these genes is expressed, as the
mRNA levels remain undetectable during embryo emergence (Fig. 2).
Seed storage proteins like -conglycinin are present in dehydrated
seeds and are degraded during the first 7-10 days after imbibition.
The hydrolysis of these proteins contributes to the energetic and
structural requirements of the rapidly growing embryo (1-3, 42). Since
the SBP mRNA is absent in newly emerging cotyledons, it was of
interest to examine whether the SBP protein was present in dehydrated
seeds and, if so, whether it was degraded after imbibition in a time
frame similar to other storage proteins. To examine these questions,
microsomal proteins from seeds at 2-7 days after imbibition were
isolated and separated by SDS-PAGE, and the levels of the SBP were
probed by immunoblotting. Fig. 3 shows that the SBP is
present in imbibed seeds. Analysis of total membrane protein extracts
from dehydrated seeds confirms the presence of the SBP in this dormant
tissue (data not shown). After imbibition, the level of SBP decreases
with kinetics similar to other storage proteins and reaches
undetectable levels 7 days after imbibition. The slight increase in the
amount of SBP at 3-4 days is probably indicative of more protein being
loaded on the gel in these lanes and does not represent de
novo synthesis, as no SBP mRNA is present in these tissues.
The smaller band at ~42 kDa represents a proteolytic product that
sometimes forms during protein extraction and SDS-PAGE. These data show
that despite the fact that the SBP is localized to the plasma membrane,
it is regulated in a manner similar to the seed storage proteins. Probing of the purified plasma membrane proteins with antibodies specific for
-conglycinin showed that no plasma membrane proteins shared antigenic sites with this storage protein (data not shown).
Oligomeric Organization of the SBP
Examination of
vicilin-like proteins using analytical ultracentrifugation or
size-exclusion chromatography shows that these proteins are organized
as stable homotrimers (43-47). In addition, the three-dimensional
structure of phaseolin and canavalin shows that the trimer is composed
of monomers arranged around a 3-fold axis of symmetry (7, 8, 10). Since
detailed structural information is available for the storage proteins
and the SBP shares strong sequence similarity with the vicilin-like
storage proteins, we examined the organization of SBP in membrane
preparations from soybean cotyledons using analytical
ultracentrifugation in the absence or presence of reducing reagents.
The results of these analyses show that under nonreducing conditions,
the SBP is part of two multimeric complexes (Fig.
4A). Using proteins of known size and
organization as standards, the molecular masses of SBP-containing complexes were determined; these findings suggest that the two complexes represent dimers (125 kDa) and trimers (190 kDa) of the SBP.
When the sample is denatured by incubation with a strong reducing
reagent (-mercaptoethanol), the multimeric complexes dissociate, and
only monomers of SBP are observed (Fig. 4A), suggesting that
interactions between subunits of this complex are mediated by disulfide
linkages. These experiments, however, fail to exclude the possibility
that other proteins may be present in these higher order complexes.
One explanation for the presence of two SBP-containing complexes is that the larger complex (190 kDa) is unstable and dissociates to form the smaller complex (125 kDa) during sample preparation. Since the SBP is present on the extracellular surface of the plasma membrane (22), treatment of soybean cotyledons with the membrane-impermeant cross-linking reagent DTSSP followed by analysis of the microsomal proteins using ultracentrifugation should address whether the larger complex is labile. The results of this experiment show that both SBP-containing complexes are observed, even when the proteins are covalently cross-linked before membrane purification (Fig. 4A). It may still be possible that the DTSSP is unable to cross-link all available SBP complexes because the intact cotyledons are relatively large and contain cell walls. Alternatively, the smaller complex may represent intermediates in the process of forming the larger complexes. The legumin-like proteins exhibit a similar accumulation of partially assembled oligomers at different times during seed development (2).
To further examine the multimeric complexes observed using ultracentrifugation, plasma membrane proteins isolated from soybean cotyledons were separated by SDS-PAGE either in the presence or absence of reducing reagent, transferred to nitrocellulose, and immunostained. Fig. 4B shows that in the presence of reducing reagent, the SBP is present as a single 62-kDa band. However, if the plasma membrane proteins are resolved in the absence of reducing reagent, the SBP is present as two slower migrating complexes with molecular masses of 125 ± 7.5 kDa and 190 ± 10 kDa (Fig. 4B). These observations are consistent with the results obtained via analytical ultracentrifugation and suggest that the SBP is associated as dimers and trimers in vivo.
Functional Divergence of a Vicilin-like Protein and [14C]Sucrose Uptake in Full-length and Truncated Forms of the SBPVicilin-like storage proteins have been identified in distantly related plant species including angiosperms and gymnosperms (36, 48). Examination of relatedness among vicilin-like storage proteins using an unrooted phylogenetic tree shows that the SBP is more closely related to the vicilin-like proteins than to the legumin-like proteins (11 S) (data not shown). The overall sequence identity between individual members of the family is highly variable (28-82%) with the SBP, sharing the highest levels of identity in the less variable region (i.e. residues 108-482) with the wheat (33%), cacao (33%), and maize (34%) sequences.
The observations that the SBP is present in dehydrated seeds and shares
significant homology with the vicilin-like seed storage proteins are
unexpected because the SBP is known to bind sucrose (as evidenced by
the photoaffinity labeling originally used to identify this protein),
is tightly associated with the plasma membrane of cells involved in
active sucrose uptake, and is able to mediate sucrose uptake when
ectopically expressed in yeast (15, 20-22). However, because the SBP
shares a high degree of sequence and, presumably, structural
conservation with vicilin-like proteins, it remains possible that other
vicilin-like proteins may also mediate sucrose uptake or that
overexpression of a vicilin-like protein in yeast may induce a cryptic
sucrose transport activity. To test these possibilities, the cDNA
encoding a closely related vicilin-like protein, glb1 from
maize, was subcloned in front of the constitutive yeast alcohol
dehydrogenase 1 (ADH1) promoter of the pMK195 yeast expression vector.
This vector was transformed into the susy7/ura3
yeast strain (15, 16), and the ability of yeast expressing the Glb1
protein to mediate [14C]sucrose uptake was determined.
The susy7/ura3 strain has the plant enzyme
sucrose synthase, a cytosolic enzyme that hydrolyzes sucrose into
UDP-glucose and fructose, stably integrated into its genome and
contains mutations in both invertase and the MALO transporter (the latter has a low affinity for sucrose (15, 16)). Fig.
5 shows that yeast expressing the full-length SBP (CG1)
are able to mediate [14C]sucrose uptake. In contrast,
yeast containing either the pMK195 vector alone (OG1) or expressing the
Glb1 protein are unable to mediate the uptake of
[14C]sucrose. These results show that sucrose uptake
mediated by the SBP does not result from the overexpression of a
vicilin-like protein in yeast and furthermore suggest that the SBP is
functionally divergent from other members of the vicilin-like protein
family.
A truncated form of the SBP has previously been shown to mediate
sucrose uptake in a proton-independent manner and not adhere to
Michaelis-Menten kinetic parameters (15). This modified SBP was a
chimeric protein consisting of residues 1-444 of the SBP and a portion
of the yeast alcohol dehydrogenase 3-noncoding region. Fig. 5 also
shows that the truncated form of the SBP (OG2) is more efficient at
mediating sucrose uptake than the full-length protein (CG1). There are
at least two interpretations of these results. First, the truncated
form may be processed more efficiently, resulting in more functional
protein arriving at the plasma membrane to mediate uptake.
Alternatively, deletion of the C-terminal 80 amino acids from the SBP
may diminish its ability to form oligomers.
The current study shows that the SBP shares significant structural
and sequence similarity with the vicilin-like seed storage proteins. In
addition, the SBP and the soybean vicilin-like storage protein,
-conglycinin, are expressed coordinately during seed development.
Although the SBP protein accumulates in developing cotyledons, the
presence of the SBP in dried, mature seeds and its rapid degradation
during germination provide additional insight into its relationship
with the vicilin-like protein family.
Sequence analysis reveals that the SBP shares several primary elements
that are conserved among members of the vicilin-like protein family.
These include a hydrophobic domain at the N terminus, a putative
vacuolar signal sequence, a hydrophilic segment enriched in charged
amino acids, and an internally repeated domain consisting of
approximately 170 residues. The three-dimensional structures of
phaseolin and canavalin demonstrate that these repeated segments form
+
structural motifs consisting of a
barrel with a Swiss roll topology followed by an extended loop containing several short
helices. Within these structural motifs, many identical or
conservatively replaced amino acids occupying equivalent positions can
be identified by aligning the primary sequences of the vicilin-like protein family (4-10). In fact, the N-terminal domain of the SBP contains 12 of 13 residues that are conserved across the vicilin-like family, the C-terminal domain contains 10 of 12 strictly conserved residues, and the intervening segments contain a strictly conserved residue (Gln-289). Each of these residues is involved in stabilizing inter- or intramolecular contacts that coordinate the three-dimensional structure of the vicilin-like molecules (8, 10).
Interestingly, there are only two residues, a Pro and a Gly, in each
+
motif that are conserved in both the N- and C-terminal domains of all proteins belonging to the vicilin-like and legumin-like families (8). The N-terminal
+
domain of the SBP contains these
highly conserved residues in proper register (Pro-163 and Gly-177,
using SBP numbering). However, as has been noted before, the C-terminal
+
domain of the SBP contains a Ile for Pro substitution
(Ile-361), but Gly-376 remains conserved (Fig. 1; Ref. 36).
Additionally, the N
-terminal
+
domain of the SBP contains an
Ala for Pro substitution (Ala-157). Both of these Pro residues
contribute to the formation of
bulges in both domains of the
canavalin and phaseolin proteins (7, 8, 10). In the case of phaseolin,
the
bulge in the C-terminal domain (49) mediates the interaction of
the J and J
sheets that flank the extended
-helical segment of
each domain. Alignment of the SBP with canavalin and phaseolin suggests
that there is a 19-residue insertion present in the primary sequence of
the SBP in this region (Fig. 1). Since the three-dimensional structure
has not been defined, the effects of this insertion remain unknown;
however, it is tempting to speculate that the Ile for Pro substitution
at position 361 in the C-terminal domain of the SBP might coordinate
the interaction of this extended portion of the protein with the
barrel. In addition, this 19-amino acid insertion in the C-terminal
domain may be important for the functional divergence of the SBP from other members of the vicilin-like protein family. Another departure from the vicilin-like proteins present in the SBP is a 5-residue insertion following residue Gln-381 that corresponds to the EF loop of
the C-terminal domain of phaseolin and canavalin (Fig. 1). This
structural comparison, then, provides insight into which residues and
domains may be altered to examine the impact on SBP-mediated sucrose
uptake.
Canavalin, phaseolin, and other vicilin-like storage proteins exist as homotrimeric complexes with molecular masses of 150-200 kDa (7, 13, 45, 46). The three monomers assemble around an exact 3-fold axis to form a toroid complex with an outside diameter of 86-88 Å and an internal hole of 18 Å (8, 10). Although the effects of the Ile (Ile-361 of the SBP) for Pro substitution and the Ala for Pro substitution at position 157 on the oligomeric organization of the SBP remains unclear, it appears that these substitutions do not prevent the SBP from forming higher order complexes. Analytical ultracentrifugation and nonreducing SDS-PAGE indicate that the SBP is present in higher order complexes of a size consistent with trimers and support the notion that the SBP is structurally related to the vicilin-like proteins. These results also indicate that the SBP may be present as dimers in vivo. In this regard, it is interesting that the C-terminal truncated form of the SBP is more efficient than the full-length in mediating sucrose uptake into yeast, as it might be expected that this deletion would interfere with the formation of oligomers. Alternatively, this difference in activity may be due to different amounts of the SBP or the truncated SBP arriving at the plasma membrane. Analyses of SBP concentration at the plasma membrane and the oligomeric form of SBP in yeast are under way to address these questions. Although the dimers observed in vivo are consistently present and cross-linking the proteins before sample preparation does not increase the trimer:dimer ratio, the significance of these dimeric complexes remains obscure. It remains possible, however, that the native oligomer is present as a trimer and that disruption of the cells results in some degradation of this higher order structure that cannot be prevented by the cross-linking reagent used, perhaps because the DTSSP is unable to effectively penetrate the entire cotyledonary tissue.
Despite the sequence and structural similarity the SBP shares with the vicilin-like proteins, it remains distinct from these seed storage proteins in several ways. First, unlike the storage proteins whose distribution is limited to the seeds (1, 2), expression of the SBP is not limited to this tissue. In fact, the SBP is expressed in leaves and is associated with the plasma membrane of cells comprising the mature phloem (21). Second, while the vicilin-like storage proteins are contained in membrane-bound protein bodies in seeds, the SBP is associated with the plasma membrane in this tissue (21, 22). This may be related to the LKKF motif found in the SBP corresponding to the LQ(R/K)F motif postulated in the processing of vicilin-like proteins through the secretory system and into vacuoles and protein bodies. Third, while the vicilin-like proteins comprise up to 30% of the protein found in seeds, the SBP is a low abundance protein comprising <0.1% of the total protein present in cotyledons.
Finally, the ability of the SBP to mediate proton-independent, relatively nonsaturable uptake of sucrose when ectopically expressed in yeast clearly distinguishes it from the vicilin-like storage proteins. It is possible that SBP-mediated sucrose uptake might result from the aberrant overexpression of a vicilin-like plant protein in yeast cells. To examine this possibility, Glb1, a vicilin-like protein from maize (28, 29), was expressed in yeast, and the capacity of the yeast to mediate [14C]sucrose uptake was assessed. The inability of yeast cells expressing Glb1 to accumulate sucrose demonstrates that a closely related vicilin-like protein is unable to mediate sucrose uptake and that SBP-mediated sucrose uptake does not result from the ectopic expression of a vicilin-like protein in yeast. It is clear, therefore, that the SBP protein is functionally distinct from the Glb1 protein, and its targeting to the plasma membrane and association with cells of the mature phloem demonstrate that the SBP has diverged significantly from other members of this family.
Since it is unlikely that the similarities between the SBP and the vicilin-like proteins at the primary level are the result of convergent evolution, it appears that the SBP belongs to a superfamily of proteins containing the globulin storage proteins and the spherulin-like proteins of Myxomycetes that share homology with the germins of cereals (36, 39, 50, 51). The evolutionary relationship between the SBP and the vicilin-like storage proteins may have important implications for elucidating the mechanism of SBP-mediated sucrose uptake. Kinetic analysis of SBP-mediated sucrose uptake in yeast indicates that this process is both proton-independent and relatively nonsaturable (15). These unique attributes closely parallel prior physiological observations of a nonsaturable sucrose uptake system in plants (19, 52, 53). Thus, the mechanism of SBP-mediated uptake is biochemically distinct from other established membrane transport mechanisms. Ko et al. (10) have suggested that the 18-Å channel formed in the canavalin trimer may play a role in the transport of solvents, metabolites, or water. They further envision the disc-shaped trimers to be stacked like plates so that the molecules form a network of solvent-filled microtubules (10). Although we do not yet know if trimers of the SBP are required for SBP-mediated sucrose uptake or whether such channels are formed by interacting SBP monomers, such a model remains an attractive hypothesis that may explain the unique mechanism of SBP-mediated sucrose uptake into plant cells. As such, the hypothesized structural relatedness of the SBP with the vicilin-like proteins provides a novel framework for probing the mechanism of sucrose uptake mediated by the SBP.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) P10562[GenBank] (canavalin) and P02853[GenBank] (phaseolin).
We thank Steve Thompson (Visualization, Analysis, and Design in the Molecular Sciences Center, Washington State University) for assistance in performing sequence analysis. The gift of glb1 cDNA and anti-Glb1 antiserum from Dr. Alan Kriz (Dekalb Plant Genetics) is gratefully acknowledged. We also thank Joerg Riesmeier, Marion Kwart, and Dr. Wolf Frommer for supplying the original yeast strain and vector.