Recombinant Arabidopsis thaliana GDT1-like protein 3 (At5g36290)
Description
Introduction to At5g36290
At5g36290, also known as GDT1-like protein 3 or AtGDT1-Like3, is encoded by a gene located on chromosome 5 of the Arabidopsis thaliana genome . This protein belongs to the UPF0016 family, which is highly conserved across eukaryotes and prokaryotes . While most non-plant eukaryotes possess only a single member of this family, plant genomes contain multiple UPF0016 proteins that differ primarily in their N-terminal regions, suggesting functional diversification within plants .
At5g36290 is one of five UPF0016 family members identified in Arabidopsis thaliana. The other members include PAM71 (PHOTOSYNTHESIS AFFECTED MUTANT71, At1g64150), PAM71-HL (At4g13590), GDT1-Like4 (At1g25520), and GDT1-Like5 (At1g68650) . Among these proteins, At5g36290 shares approximately 38% sequence identity with PAM71, the founding member of the UPF0016 family in plants .
Primary Structure and Domains
The mature form of At5g36290 protein consists of 268 amino acids (residues 26-293) . Its primary sequence is highly conserved and characterized by the presence of specific motifs that are hallmarks of the UPF0016 family . The complete amino acid sequence of the mature protein is:
QDSVVENNERQESEGSGKELGRRGMVGTERIGVDTVVDNIGALGLNLDLDATAPSVFDAL FSSFSMILVTEIGDETFIIAALMAMRHPKATVLSGALSALFVMTILSTGLGRIVPNLISR KHTNSAATVLYAFFGLRLLYIAWRSTDSKSNQKKEMEEVEEKLESGQGKTPFRRLFSRFC TPIFLESFILTFLAEWGDRSQIATIALATHKNAIGVAIGASIGHTVCTSLAVVGGSMLAS RISQRTVATVGGLLFLGFSVSSYFYPPL
Membrane Topology
Like other members of the UPF0016 family, At5g36290 is predicted to contain six transmembrane domains arranged in two clusters of three (TM1-TM3 and TM4-TM6), separated by a central loop . This structural arrangement is highly conserved across the UPF0016 family and is believed to be essential for their function as cation transporters .
A distinctive feature of At5g36290 is the presence of two highly conserved E-x-G-D-(KR)-(TS) motifs located in transmembrane domains TM1 and TM4 . These motifs contain negatively charged acidic residues that likely provide a suitable environment for binding and transporting cations such as manganese (Mn²⁺) or calcium (Ca²⁺) .
N-terminal Region
One of the key features distinguishing At5g36290 from other Arabidopsis UPF0016 family members is its N-terminal region. Unlike PAM71 and PAM71-HL, which possess chloroplast transit peptides, At5g36290 is predicted to contain an N-terminal cleavable secretory pathway signal peptide . This structural difference likely accounts for the distinct subcellular localization and function of At5g36290 compared to its family members.
Subcellular Localization
Based on subcellular prediction analyses, At5g36290 is predominantly localized to the Golgi apparatus . This localization pattern is consistent with the presence of its N-terminal secretory pathway signal peptide . The high confidence score (1.000) for Golgi localization reported in the SUBA5 database strongly supports this prediction .
The Golgi localization of At5g36290 mirrors that of its orthologs in other eukaryotes, such as GDT1 (GCR1 DEPENDENT TRANSLATION FACTOR 1) in yeast and TMEM165 (TRANSMEMBRANE PROTEIN 165) in humans, both of which are also Golgi-resident proteins . This conservation of subcellular localization across diverse eukaryotes suggests a fundamental role for these proteins in Golgi function.
Putative Function as a Cation Transporter
Although the precise function of At5g36290 has not been fully characterized, its structural similarity to other UPF0016 family members suggests a role in cation transport . Studies on related proteins, particularly the yeast GDT1 and human TMEM165, have demonstrated their involvement in calcium and manganese transport .
Research has shown that GDT1 in yeast functions as both a calcium and manganese transporter localized to the Golgi apparatus . Similarly, TMEM165 in humans plays a crucial role in Golgi manganese homeostasis, which is essential for proper protein glycosylation . Defects in TMEM165 cause a rare inherited disease called Congenital Disorder of Glycosylation type II (CDG-II), characterized by severe growth and psychomotor retardations .
Evolutionary Conservation
The UPF0016 family is widely distributed across eukaryotes and prokaryotes, indicating its ancient evolutionary origin and fundamental importance in cellular function . While most eukaryotes possess only a single member of this family, plants have evolved multiple UPF0016 proteins with different subcellular localizations .
In Arabidopsis thaliana, the five UPF0016 family members show distinct patterns of subcellular localization: PAM71 is targeted to the thylakoid membrane, PAM71-HL to the chloroplast envelope, and At5g36290 likely to the Golgi apparatus . GDT1-Like4 and GDT1-Like5, which lack N-terminal extensions, are also predicted to enter the secretory pathway .
Functional Diversification
The diversification of UPF0016 proteins in plants suggests an adaptation to meet the specialized cation transport needs of different cellular compartments . While PAM71 has been demonstrated to function as a manganese transporter essential for photosynthesis, the specific roles of the other Arabidopsis UPF0016 members, including At5g36290, remain to be fully elucidated .
The presence of At5g36290 in the Golgi apparatus, similar to non-plant UPF0016 members, suggests that it may perform functions analogous to those of yeast GDT1 and human TMEM165 . These functions could include maintaining proper calcium and manganese levels in the Golgi lumen, which are critical for various cellular processes, including protein glycosylation .
Expression and Purification
The recombinant form of At5g36290 has been successfully produced using Escherichia coli as an expression system . Specifically, the mature protein (amino acids 26-293) has been expressed with an N-terminal histidine (His) tag to facilitate purification . The resulting recombinant protein has been purified to greater than 90% homogeneity, as determined by SDS-PAGE analysis .
Physical and Chemical Properties
The recombinant At5g36290 protein is typically supplied as a lyophilized powder and can be reconstituted in deionized sterile water to achieve concentrations of 0.1-1.0 mg/mL . For long-term storage, the addition of 5-50% glycerol (with 50% being the default final concentration) and storage at -20°C/-80°C is recommended . Repeated freeze-thaw cycles should be avoided to maintain protein integrity .
The reconstituted protein is stored in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . Working aliquots can be stored at 4°C for up to one week . These properties are important considerations for researchers working with the recombinant protein in experimental settings.
Model for Understanding Cation Transport
The availability of recombinant At5g36290 provides researchers with a valuable tool for investigating the molecular mechanisms of cation transport in plant cells . By studying the structure-function relationships of this protein, researchers can gain insights into how plants maintain proper calcium and manganese homeostasis in different cellular compartments.
Comparative Studies with Human and Yeast Orthologs
Research on At5g36290 can also contribute to our understanding of its orthologs in other organisms, particularly the human TMEM165 . Given that mutations in TMEM165 cause the rare genetic disorder CDG-II, studies on the plant protein may provide indirect insights into the molecular basis of this disease .
Some research groups have used yeast as a model organism to study the function of GDT1 (the yeast ortholog) and, by extension, to understand the role of TMEM165 in human cells . This comparative approach across different organisms can help elucidate conserved mechanisms of cation transport and their implications for cellular physiology and disease.
Functional Characterization
Despite the structural information available for At5g36290, its precise physiological function remains to be fully elucidated. Future research should focus on determining whether this protein indeed functions as a cation transporter and, if so, its specificity for different cations such as calcium and manganese.
Regulation and Interaction Partners
Another important area for future investigation is the regulation of At5g36290 expression and activity, as well as its potential interaction with other proteins. Understanding these aspects would provide insights into how this protein is integrated into the broader cellular network controlling cation homeostasis.
Role in Plant Physiology and Development
The physiological significance of At5g36290 in plant growth, development, and stress responses remains largely unexplored. Studies using loss-of-function or overexpression approaches could help determine the importance of this protein for various aspects of plant physiology under different environmental conditions.
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will prepare the product according to your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life for the liquid form is 6 months at -20°C/-80°C. The shelf life for the lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What is the molecular function of GDT1-like protein 3 in Arabidopsis thaliana?
GDT1-like protein 3 (At5g36290), also known as PML3 or BICAT3, belongs to the evolutionarily conserved UPF0016 family. It functions as a Ca²⁺/Mn²⁺ antiporter localized to the Golgi apparatus, where it mediates Mn²⁺ uptake essential for protein glycosylation processes. The protein contains two conserved E-Φ-G-D-(KR)-(TS) motifs characteristic of the UPF0016 family transporters, which are critical for its transport activity. Functionally, it is analogous to human TMEM165 and yeast GDT1 proteins, suggesting evolutionary conservation of this transport mechanism across diverse organisms.
How does GDT1-like protein 3 differ from other members of the GDT1 family?
While GDT1-like protein 3 (At5g36290) localizes to the Golgi apparatus and functions primarily in manganese transport for glycosylation, other family members have distinct localizations and functions:
The GDT1-like protein 1 (At1g64150) is chloroplast-localized and functions as a Ca²⁺/H⁺ antiporter, playing a critical role in photosynthesis (hence its alternative name PHOTOSYNTHESIS AFFECTED MUTANT71) . In contrast, the yeast ortholog Gdt1p has been directly shown to transport protons across biological membranes in exchange for Ca²⁺ and Mn²⁺ .
What phenotypes are associated with GDT1-like protein 3 deficiency?
GDT1-like protein 3 deficiency results in several observable phenotypes related to manganese homeostasis. Knockout mutants (pml3-1, pml3-2) exhibit characteristic manganese-deficiency phenotypes, including leaf curling and chlorosis, particularly under low manganese conditions. At the cellular level, the protein is essential for pollen tube tip growth by enabling proper pectin deposition in cell walls. Notably, complete null mutants of At5g36290 display embryonic lethality, highlighting its indispensable role in plant development. These phenotypes underscore the critical importance of GDT1-like protein 3 in maintaining proper manganese homeostasis for essential cellular processes.
How can I design experiments to characterize the transport kinetics of GDT1-like protein 3?
When designing experiments to characterize the transport kinetics of GDT1-like protein 3, consider implementing these methodological approaches:
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Reconstitution in proteoliposomes: Purify the recombinant protein (>90% purity achievable through His-tag affinity chromatography) and reconstitute it into liposomes with fluorescent Ca²⁺ or Mn²⁺ indicators. This system allows for controlled manipulation of ion gradients across membranes.
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pH-dependent transport assays: Similar to experiments with yeast Gdt1p, measure H⁺ transport using pH-sensitive fluorescent dyes when exposing the reconstituted protein to different Ca²⁺ or Mn²⁺ concentrations . The yeast ortholog experiments showed that Gdt1p-expressing cells acidify more than control cells when exposed to identical H⁺ gradients, providing direct evidence of proton transport capability .
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Radioisotope flux analysis: Use ⁴⁵Ca²⁺ and ⁵⁴Mn²⁺ tracers to quantify transport rates under various conditions.
An example data collection table would be:
| Condition | Initial Rate (nmol/min/mg) | Km (μM) | Vmax (nmol/min/mg) | Hill Coefficient |
|---|---|---|---|---|
| pH 6.0 | ||||
| pH 6.5 | ||||
| pH 7.0 | ||||
| pH 7.5 |
Record measurements at different substrate concentrations for each pH condition to determine Michaelis-Menten kinetics parameters.
What approaches are recommended for investigating the structure-function relationship of conserved motifs in GDT1-like protein 3?
The GDT1-like protein 3 contains two conserved E-Φ-G-D-(KR)-(TS) motifs that are characteristic of UPF0016 family transporters and likely crucial for transport function. To investigate structure-function relationships:
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Site-directed mutagenesis: Create point mutations in key residues of the conserved motifs. This approach was successfully employed with SDA1 (another defense-associated protein), where mutations in the SWAD domain (D8A and Q9R) significantly reduced PR1 expression, demonstrating the domain's functional importance .
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Complementation assays: Express mutated versions in knockout plants to assess functional rescue. Analyze phenotypes including:
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Pollen tube growth rates
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Manganese deficiency symptoms
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Glycosylation patterns
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Transport activity assays: Compare wild-type and mutant protein transport activities using reconstituted proteoliposomes.
A systematic experimental design might include:
| Mutation | Position | Conserved Motif | Expected Effect | Actual Effect |
|---|---|---|---|---|
| E→A | First motif | E-Φ-G-D-(KR)-(TS) | Disrupted cation binding | |
| G→A | First motif | E-Φ-G-D-(KR)-(TS) | Altered protein structure | |
| D→N | First motif | E-Φ-G-D-(KR)-(TS) | Neutralized charge | |
| E→A | Second motif | E-Φ-G-D-(KR)-(TS) | Disrupted cation binding |
Similar to studies with SDA1, RT-qPCR could be used to quantify expression levels of the mutant constructs to ensure comparable expression levels .
How can I investigate the role of GDT1-like protein 3 in plant stress responses?
Investigating GDT1-like protein 3's role in stress responses requires multi-layered experimental approaches:
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Expression profiling: Monitor At5g36290 expression under various stress conditions (drought, salt, heat, pathogen infection) using RT-qPCR. This approach was effectively used to study SDA1 expression following pathogen infection, revealing its role in defense signaling .
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Stress tolerance phenotyping: Compare wild-type and knockout/knockdown plants under stress conditions, measuring:
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Relative growth rate
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Photosynthetic efficiency
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Stress marker gene expression
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Ion accumulation profiles
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Genetic interaction studies: Create double mutants with known stress response pathway components to identify epistatic relationships. This strategy revealed that SDA1 functions downstream of PAD4, EDS1, GDG1, and NDR1 but upstream or independent of SID2 and NPR1 in pathogen defense pathways .
Data could be organized in stress response comparison tables:
| Stress Type | Parameter | Wild-type | GDT1-like protein 3 Mutant | Complemented Line |
|---|---|---|---|---|
| Manganese deficiency | Chlorophyll content (μg/g FW) | |||
| Manganese deficiency | Root growth (cm) | |||
| Salt stress | Survival rate (%) | |||
| Pathogen infection | PR gene expression |
This approach would provide comprehensive insights into the protein's role in stress response networks.
What are the optimal conditions for recombinant expression and purification of GDT1-like protein 3?
The optimal conditions for recombinant expression and purification of GDT1-like protein 3 have been established through previous studies:
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Expression system: Escherichia coli using codon-optimized expression vectors has proven effective. The recombinant protein corresponds to residues 26-293 of the full-length Arabidopsis GDT1-like protein 3, with an N-terminal 10× His tag to facilitate purification.
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Purification protocol:
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Affinity chromatography via the His tag yields >90% purity as determined by SDS-PAGE
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Consider using immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography
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Buffer conditions:
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Storage buffer: Tris/PBS-based buffer containing 6% trehalose at pH 8.0
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For reconstitution: Use deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol (final concentration)
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Storage recommendations:
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Store at -20°C/-80°C upon receipt
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Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles
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Working aliquots can be stored at 4°C for up to one week
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A similar approach was successfully used for GDT1-like protein 1 (At1g64150), which was expressed in E. coli with an N-terminal His tag .
What methods can I use to verify the functional activity of purified GDT1-like protein 3?
To verify the functional activity of purified GDT1-like protein 3, implement these approaches:
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Proteoliposome-based transport assays:
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Reconstitute the purified protein into liposomes
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Measure Mn²⁺ or Ca²⁺ uptake using fluorescent indicators or radioisotopes
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Compare transport rates with control liposomes lacking the protein
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pH gradient experiments: Following the methodology used for yeast Gdt1p, monitor internal pH changes over time in response to ion gradients . The experiment should reveal:
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Greater cytosolic acidification in GDT1-expressing systems compared to controls when exposed to identical proton gradients
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Differential final intracellular pH when applying identical [H⁺] gradients
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Complementation assays: Test whether the purified protein can rescue phenotypes in knockout mutants when delivered to cellular systems.
For pH gradient experiments, data could be recorded as follows:
| Time (min) | Control pH | GDT1-like protein 3 pH | Difference | P-value |
|---|---|---|---|---|
| 0 | ||||
| 5 | ||||
| 10 | ||||
| 15 | ||||
| 20 |
Similar to yeast Gdt1p studies, a proper functional GDT1-like protein 3 would show statistically significant differences in pH measurements compared to controls .
How can I design effective gene expression studies to understand GDT1-like protein 3 regulation?
When designing gene expression studies for GDT1-like protein 3, consider these methodological approaches:
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Temporal expression analysis: Monitor expression at different developmental stages and in response to environmental stimuli using RT-qPCR. This approach was effectively used to study SDA1 expression patterns, revealing its rapid induction following pathogen infection .
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Tissue-specific expression: Use promoter-reporter fusions (e.g., GDT1p:GUS) to visualize expression patterns across tissues.
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Expression in mutant backgrounds: Similar to the analysis performed for SDA1, examine At5g36290 expression in various signaling pathway mutants to establish regulatory hierarchies . For SDA1, this approach revealed that its expression was compromised in pad4, eds1, pbs3/gdg1/win3, and ndr1 mutants, but not in sid2 and npr1 mutants .
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Transcription factor binding analysis: Perform chromatin immunoprecipitation (ChIP) to identify transcription factors that regulate At5g36290 expression.
For RT-qPCR analysis, prepare data tables as follows:
| Treatment/Condition | Time Point | Relative Expression (Fold Change) | Statistical Significance |
|---|---|---|---|
| Control | 0h | 1.0 | - |
| Mn deficiency | 6h | ||
| Mn deficiency | 12h | ||
| Mn deficiency | 24h | ||
| Ca deficiency | 6h | ||
| Ca deficiency | 12h | ||
| Ca deficiency | 24h |
This methodical approach will provide comprehensive insights into the regulatory mechanisms controlling GDT1-like protein 3 expression in different contexts3 .
What experimental controls are critical when studying manganese transport by GDT1-like protein 3?
When studying manganese transport by GDT1-like protein 3, implement these critical experimental controls:
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Empty vector controls: For expression studies, include systems transformed with empty vectors to account for background effects and establish baseline transport activity.
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Inactive mutant controls: Generate transport-deficient mutants (through site-directed mutagenesis of conserved residues) to serve as negative controls. This approach is similar to the strategy used for studying SDA1, where mutations in the SWAD domain created functionally compromised versions of the protein .
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Ion specificity controls: Include assays with other divalent cations (Zn²⁺, Fe²⁺, Cu²⁺) to confirm specificity for Mn²⁺ and Ca²⁺.
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Membrane integrity controls: Monitor membrane potential and integrity to ensure that observed ion fluxes are protein-mediated rather than due to membrane leakage.
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pH controls: Since GDT1 proteins function as antiporters with H⁺, maintain consistent pH conditions and include pH monitoring. As demonstrated with yeast Gdt1p, pH affects transport activity, with greater cytosol acidification observed in GDT1-expressing cells compared to control cells under identical conditions .
When creating data tables, ensure proper control inclusion:
| Sample | Mn²⁺ Transport Rate | Ca²⁺ Transport Rate | H⁺ Exchange Rate | Membrane Potential |
|---|---|---|---|---|
| Wild-type GDT1-like protein 3 | ||||
| E→A mutant | ||||
| Empty vector | ||||
| + Ionophore A23187 | ||||
| + Protonophore CCCP |
This comprehensive control strategy will enable robust interpretation of transport data and minimize experimental artifacts .
