Sucrose Synthase: Expanding Protein Function

Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. This inference is based on the following observations: (a) tissue-specific, isoform-dependent and metabolically-regulated association of SUS with mitochondria and (b) isoform-specific and anoxia-responsive interaction of SUS with the voltage-dependent anion channel (VDAC), the major outer mitochondrial membrane protein. More recent work shows that both VDAC and SUS are also localized to the nucleus in maize seedling tissues. Their intricate regulation under anoxia indicates that these two proteins may have a role in inter-compartmental signaling.Key Words: sucrose synthase, mitochondria, nucleus, localization, voltage-dependent anion channel (VDAC), non-catalytic rolesThe biochemical function of a protein is encoded within its primary sequence and can often be deciphered by simple in vitro assays. The cellular or organismal function of a protein is frequently the same as its biochemical activity. However, for many proteins, the biological function cannot be easily derived based on its biochemical function. This appears to be particularly true when the gene encoding the protein has a history of duplication and is represented by a family of paralogs. In maize and other species, sucrose synthase (SUS) isoforms are almost identical in their catalytic properties.^1,2 However, the characteristic phenotypes of mutants in specific isoforms suggest that the isoforms contribute to vastly different organismal functions.^2–4 Our interest is to identify the range of functions that maize SUS isoforms may have and elucidate the molecular basis of this functional diversity. Although expression divergence and consequent variation in their cellular abundance significantly contributes to this diversity,⁵ other factors such as intracellular distribution, post-translational modifications and interacting partners,^3,4,6,7 seem to be equally critical for the functional diversification of different SUS isoforms.Our study, spurred by a bioinformatics prediction, opened up a new facet of SUS biology, in that the protein may have organelle-based functions.⁸ Our analysis indicated that two of the three maize SUS isoforms (SH1 and SUS1) partly localize to mitochondria and nuclei, compartments not related to sucrose metabolism. In addition to this isoform-specificity, the compartmentation of SUS isoforms is influenced by developmental as well as environmental cues. Furthermore, its isoform-specific interaction with the voltage-dependent anion channel (VDAC) and an apparent conservation of SUS mitochondrial targeting across plant species suggest that SUS may have novel, noncatalytic biological functions. Our recent work shows that along with SUS, VDAC is also localized to the nucleus and these two proteins are inversely regulated in these two compartments under anoxic stress, indicating SUS-VDAC interaction may play a role in inter-compartmental signaling (Fig. 1).Open in a separate windowFigure 1Current working model of SUS-VDAC interactions in maize root tip cells. Prolonged anoxia leads to de-oligomerization of VDAC and the release of SUS from mitochondria, resulting in the migration of SUS to the nucleus. We hypothesize that the nuclear accumulation of SUS signals the induction of cell death pathway leading to the death of the root tip in anoxic maize seedlings. The insets show the primary root tip and a part of the axis from aerobic and anoxic seedlings. The root tip death is indicated by Evans Blue staining pattern of the anoxic root. ≠ = SUS. □ = VDAC.SUS mitochondrial localization also provided us an opportunity to reinterpret the phylogeny of sucrose metabolism. The proposed origin of sucrose metabolism is equivocal between the proteobacterial and cyanobacterial lineages.^9,10 Our discovery of SUS inside mitochondria, absence of plastid-bound SUS or plastid-targeting information in any of the plant SUS proteins and occurrence of mitochondrial targeting information in proteobacterial SUS orthologs strongly support a proteobacterial origin of plant sucrose synthases.⁸ Based on a genome-wide analysis of E. coli proteins, Lucattini et al.¹¹ proposed that mitochondrial targeting information may have been derived from the preexisting sequences of the endosymbiont proteins. We hypothesize that, in addition to the structural features needed for mitochondrial association, the functional basis of SUS-VDAC interaction may have been recruited by plants from the prokaryotic SUS genes. Based on striking similarities between bacterial and mitochondrial porins in their structure as well as regulation by purine nucleotides and their role in the host-cell death as modulated by cellular ATP levels, Frade and Michaelidis¹² speculated that the eukaryotic programmed cell death may have been a consequence of acquiring aerobic metabolism via the endosymbiotic process. Is organellar SUS a part of this acquisition?     

Cytoskeleton-associated, carbohydrate-metabolizing enzymes in maize identified by yeast two-hybrid screening

We have used yeast two-hybrid screens and biochemical methods to identify glycolytic enzymes that interact with subcellular structures in hypoxic maize seedlings. As binding domain-bait fusion constructs, we have cloned actin, cytosolic aldolase, the three sucrose synthase (SUS) isoforms SUS1, SUS3, and SH1 as well as the SNF1-related protein kinase into yeast and identified cytosolic isoforms of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase, tubulin, and mitochondrial porin voltage-dependent anion channel protein (VDAC) as well as protein kinases and proteins involved in ubiquitinylation and proteasome-linked degradation as interacting activation domain-prey clones. The results were further confirmed using overlay blots (VDAC) as well as co-polymerization and co-precipitation assays (tubulin and actin). Some results were obtained that support the idea of metabolite and modification effects on the association, namely guanosine triphosphate (GTP)/MgCl₂ was necessary for the binding of enolase to actin. GAPDH is inactivated upon association with tubulin but then serves to stabilize the microtubules. The findings support the idea of the dynamic formation of locally associated complexes of enzymes involved in sucrose breakdown and glycolysis in plant cells depending on their metabolic state.     

Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1

Sucrose (Suc) synthase (SUS) cleaves Suc to form UDP glucose and fructose, and exists in soluble and membrane-associated forms, with the latter proposed to channel UDP glucose to the cellulose-synthase complex on the plasma membrane of plant cells during synthesis of cellulose. However, the structural features responsible for membrane localization and the mechanisms regulating its dual intracellular localization are unknown. The maize (Zea mays) SUS1 isoform is likely to have the intrinsic ability to interact directly with membranes because we show: (1) partial membrane localization when expressed in Escherichia coli, and (2) binding to carbonate-stripped plant microsomes in vitro. We have undertaken mutational analyses (truncations and alanine substitutions) and in vitro microsome-binding assays with the SUS1 protein to define intrinsic membrane-binding regions and potential regulatory factors that could be provided by cellular microenvironment. The results suggest that two regions of SUS1 contribute to membrane affinity: (1) the amino-terminal noncatalytic domain, and (2) a region with sequence similarity to the C-terminal pleckstrin homology domain of human pleckstrin. Alanine substitutions within the pleckstrin homology-like domain of SUS1 reduced membrane association in E. coli and with plant microsomes in vitro without reducing enzymatic activity. Microsomal association of wild-type SUS1 displayed cooperativity with SUS1 protein concentration and was stimulated by both lowering the pH and adding Suc. These studies offer insight into the molecular level regulation of SUS1 localization and its participation in carbon partitioning in plants. Moreover, transgenics with active SUS mutants altered in membrane affinity may be of technological utility.     

A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips

In order to understand the role of sucrose synthase (SuSy) in carbon partitioning, metabolic fluxes were analyzed in maize root tips of a double mutant of SuSy genes, sh1 sus1 and the corresponding wild type, W22. [U-¹⁴C]-glucose pulse labeling experiments permitted the quantification of unidirectional fluxes into sucrose, starch and cell wall polysaccharides. Isotopic steady-state labeling with [1-¹³C]-, [2-¹³C]- or [U-¹³C]-glucose followed by the quantification by ¹H-NMR and ¹³C-NMR of enrichments in carbohydrates and amino acids was also performed to determine 29 fluxes through central metabolism using computer-aided modeling. As a consequence of the suppression of SUS1 and SH1 isozymes, maize root tips diameter was significantly decreased and respiratory metabolism reduced by 30%. Our result clearly established that, in maize root tips, starch is produced from ADP-Glc synthesized in the plastid and not in the cytosol by sucrose synthase. Unexpectedly, the flux of cell wall synthesis was increased in the double mutant. This observation indicates that, in maize root tips, SH1 and SUS1 are not specific providers for cellulose biosynthesis.     

Sucrose synthase oligomerization and F-actin association are regulated by sucrose concentration and phosphorylation

Sucrose synthase (SUS) is a key enzyme in plant metabolism, as it serves to cleave the photosynthetic end-product sucrose into UDP-glucose and fructose. SUS is generally assumed to be a tetrameric protein, but results in the present study suggest that SUS can form dimers as well as tetramers and that sucrose may be a regulatory factor for the oligomerization status of SUS. The oligomerization of SUS may also affect the cellular localization of the protein. We show that sucrose concentration modulates the ability of SUS1 to associate with F-actin in vitro and that calcium-dependent protein kinase-mediated phosphorylation of recombinant SUS1 at the Ser15 site is a negative regulator of its association with actin. Although high sucrose concentrations and hyperphosphorylation have been shown to promote SUS association with the plasma membrane, we show that the opposite is true for the SUS-actin association. We also show that SUS1 has a unique 28 residue coiled-coil domain that does not appear to play a role in oligomerization, but may prove to be significant in the future for interactions of SUS with other proteins. Collectively, these results highlight the multifaceted nature of SUS association with cellular structures.     

菊芋蔗糖代谢相关产物与关键酶基因对高温的响应

蔗糖是一类重要的碳水化合物,其代谢与植物生长发育及抵抗胁迫等有密切的关系。蔗糖合成酶(SUS)、蔗糖磷酸合成酶(SPS)与蔗糖转化酶(INV)是参与蔗糖代谢的三类关键酶。本研究依据转录组测序数据,从能源植物菊芋中鉴定了2个SUS、2个SPS和7个INV基因(GenBank No:MK386943-53)。生物信息学分析表明,菊芋SUS、SPS和INV的氨基酸序列与其他物种具有较高的相似性,均属于亲水性蛋白。在25、30°C处理10、15、20 d的菊芋幼苗叶片中,这三种基因家族成员呈现不同的表达模式;除可溶性总糖含量减少外,果糖、蔗糖、蔗果三糖等含量没有发生明显变化。表明高温下幼苗蔗糖代谢关键酶基因发生了响应,蔗糖代谢处于平衡状态,显示了菊芋对高温的良好耐受性。     

Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment

Background

Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress.

Results

We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used.

Conclusions

We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.     

Noncatalytic nucleotide binding sites: Properties and mechanism of involvement in ATP synthase activity regulation

ATP synthases (F_oF₁-ATPases) of chloroplasts, mitochondria, and bacteria catalyze ATP synthesis or hydrolysis coupled with the transmembrane transfer of protons or sodium ions. Their activity is regulated through their reversible inactivation resulting from a decreased transmembrane potential difference. The inactivation is believed to conserve ATP previously synthesized under conditions of sufficient energy supply against unproductive hydrolysis. This review is focused on the mechanism of nucleotide-dependent regulation of the ATP synthase activity where the so-called noncatalytic nucleotide binding sites are involved. Properties of these sites varying upon free enzyme transition to its membrane-bound form, their dependence on membrane energization, and putative mechanisms of noncatalytic site-mediated regulation of the ATP synthase activity are discussed.     

Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves.

The serine-170 (S170) calcium-dependent protein kinase phosphorylation site of maize (Zea mays L.) sucrose synthase (SUS) (EC 2.4.1.13) has been implicated in the post-translational regulation of SUS protein stability. To clarify the proteolytic process and the role of phosphorylation, SUS degradation and proteasome activities were studied in the maize leaf elongation zone. Size-exclusion chromatography resolved two peaks of proteasome-like proteolytic activity. The large molecular mass ( approximately 1350 kDa) peak required Mg(2+) and ATP for maximal activity and was inhibited by the proteasome inhibitors MG132 and NLVS. Anion-exchange chromatography resolved a similar proteolytic activity that was activated by ATP, characteristics that are consistent with those of a 26S-proteasome. Appropriately, immunoblotting revealed the presence of a 26S-proteasome subunit and highly ubiquitinated proteins within the active fractions eluted from both columns. The smaller molecular mass ( approximately 600 kDa) peak represented only 40% of the total proteasome-like activity and is likely a maize 20S-proteasome as it was activated in vitro by low levels of sodium dodecyl sulfate (SDS). S170 phosphorylated SUS (pS170-SUS) was detected as both high molecular mass (HMM) forms and proteolytic fragments that co-eluted with 26S-proteasome activities on both size-exclusion and anion-exchange columns. Conditions that maintained maximal 26S-proteasome activity reduced the amounts of pS170-SUS recovered. In vitro, the 26S-proteasome degraded SUS and proteasome-specific inhibitors reduced SUS proteolysis. HMM-SUS conjugates were produced in vitro and immunoprecipitations suggested that some SUS might be ubiquitinated in vivo. The results suggest that S170 phosphorylation promotes the formation of HMM, ubiquitin-SUS conjugates that can be targeted for 26S-proteasome-dependent degradation.     

Mouse transition protein 1 is translationally regulated during the postmeiotic stages of spermatogenesis

Transition protein 1 (TP1) is a small basic nuclear protein that functions in chromatin condensation during spermatogenesis in mammals. Here, recently identified cDNA clones encoding mouse transition protein 1(mTP1) were used to characterize the expression of the mTP1 mRNA during spermatogenesis. Southern blot analysis demonstrates that there is a single copy of the gene for transition protein 1 in the mouse genome. Northern blot analysis demonstrates that mTP1 mRNA is a polyadenylated mRNA approximately 600 bases long, which is first detected at the round spermatid stage of spermatogenesis. mTP1 mRNA is not detectable in poly(A)+ RNAs isolated from mouse brain, kidney, liver, or thigh muscle. mTP1 mRNA is translationally regulated in that it is first detected in round spermatids, but no protein product is detectable until approximately 3 days later in elongating spermatids. In total cellular RNA isolated from stages in which mTP1 is synthesized, the mTP1 mRNA is present as a heterogeneous class of mRNAs that vary in size from about 480 to 600 bases. The shortened, heterogeneous mTP1 mRNAs are found in the polysome region of sucrose gradients, while the longer, more homogeneous mTP1 mRNAs are present in the postmonosomal fractions.     

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.     

Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity

Sucrose synthase (SUS) is phosphorylated on a major, amino-terminal site located at Ser-15 (S15) in the maize (Zea mays) SUS1 protein. Site- and phospho-specific antibodies against a phosphorylated S15 (pS15) peptide allowed direct analysis of S15 phosphorylation in relation to membrane association. Immunoblots of the maize leaf elongation zone, divided into 4-cm segments, demonstrated that the abundance of soluble (s-SUS) and membrane (m-SUS) SUS protein showed distinct positional profiles. The content of m-SUS was maximal in the 4- to 8-cm segment where it represented 9% of total SUS and occurred as a peripheral membrane protein. In contrast, s-SUS was highest in the 12- to 16-cm segment. Relative to s-SUS, m-SUS was hypophosphorylated at S15 in the basal 4 cm but hyperphosphorylated in apical segments. Differing capabilities of the anti-pS15 and anti-S15 peptide antibodies to immunoprecipitate SUS suggested that phosphorylation of S15, or exposure of unphosphorylated SUS to slightly acidic pH, altered the structure of the amino terminus. These structural changes were generally coincident with the increased sucrose cleavage activity that occurs at pH values below 7.5. In vitro S15 phosphorylation of the S170A SUS protein by a maize calcium-dependent protein kinase (CDPK) significantly increased sucrose cleavage activity at low pH. Collectively, the results suggest that (1) SUS membrane binding is controlled in vivo; (2) relative pS15 content of m-SUS depends on the developmental state of the organ; and (3) phosphorylation of S15 affects amino-terminal conformation in a way that may stimulate the catalytic activity of SUS and influence membrane association.     

Proteomic identification of ZO-1/2 as a novel scaffold for Src/Csk regulatory circuit

To elucidate the regulatory mechanism of cell transformation induced by c-Src tyrosine kinase, we performed a proteomic analysis of tyrosine phosphorylated proteins that interact with c-Src and/or its negative regulator Csk. The c-Src interacting proteins were affinity-purified from Src transformed cells using the Src SH2 domain as a ligand. LC-MS/MS analysis of the purified proteins identified general Src substrates, such as focal adhesion kinase and paxillin, and ZO-1/2 as a transformation-dependent Src target. The Csk binding proteins were analyzed by a tandem affinity purification method. In addition to the previously identified Csk binding proteins, including Cbp/PAG, paxillin, and caveolin-1, we found that ZO-1/2 could also serve as a major Csk binding protein. ZO-2 was phosphorylated concurrently with Src transformation and specifically bound to Csk in a Csk SH2 dependent manner. These results suggest novel roles for ZO proteins as Src/Csk scaffolds potentially involved in the regulation of Src transformation.     

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.     

Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me)

ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36-70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1-35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable alpha-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106-715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.