Recombinant Yarrowia lipolytica Golgi apparatus membrane protein TVP23 (TVP23)
Description
Introduction to TVP23 and Yarrowia lipolytica
Yarrowia lipolytica has gained significant attention in biotechnological research due to its remarkable protein secretion capacity and sophisticated secretory pathway system. This non-conventional yeast species has become increasingly important for the production of complex enzymes and biopharmaceuticals, as it possesses the ability to form post-translational modifications and an inherent quality control system within the endoplasmic reticulum . The Golgi apparatus, a central organelle in the secretory pathway, houses specialized proteins that facilitate the proper trafficking and modification of proteins destined for secretion or cellular compartmentalization. TVP23 represents one such specialized membrane protein located in the Golgi apparatus of Y. lipolytica, where it participates in the intricate machinery of vesicular transport .
The TVP23 protein belongs to a family that is conserved from yeast to humans, indicating its fundamental importance in cellular function across diverse species . This evolutionary conservation suggests that TVP23 mediates core processes in vesicular trafficking that have been maintained throughout eukaryotic evolution. In Y. lipolytica specifically, TVP23 contributes to the advanced secretory capabilities that make this organism valuable for various biotechnological applications, including recombinant protein production and metabolic engineering for biofuels and natural products .
The study of TVP23 not only enhances our understanding of fundamental cellular processes but also provides insights that can be leveraged to optimize Y. lipolytica for industrial applications. Researchers have developed various genetic and molecular tools to facilitate investigations of Y. lipolytica proteins, including TVP23, enabling fluorescent tagging for localization studies and targeted overexpression for functional analyses . These advances have positioned Y. lipolytica as an increasingly important model organism for both basic research and applied biotechnology.
Expression and Production of Recombinant TVP23
Commercial sources offer recombinant Y. lipolytica TVP23 with various modifications to suit different research applications. These preparations typically provide the full-length protein (amino acids 1-180) fused to an N-terminal His tag, expressed in E. coli . Following production, the protein is purified to greater than 90% homogeneity as determined by SDS-PAGE analysis and lyophilized for stability during storage .
The availability of high-quality recombinant TVP23 enables a range of experimental approaches, from structural studies to functional investigations of its role in vesicular trafficking. Proper handling and reconstitution of recombinant TVP23 are essential for maintaining its native-like properties. Manufacturers typically recommend reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of glycerol (5-50% final concentration) for long-term storage at -20°C/-80°C .
| Property | Specification |
|---|---|
| Protein Name | Golgi apparatus membrane protein TVP23 |
| Organism | Yarrowia lipolytica (strain CLIB 122 / E 150) |
| Family | TVP23 family |
| Length | 180 amino acids |
| Molecular Mass | 20.4 kDa |
| Expression System | E. coli |
| Affinity Tag | N-terminal His tag |
| Purity | >90% by SDS-PAGE |
| Form | Lyophilized powder |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| UniProt ID | Q6C7V5 |
Insights from TVP23 Homologs in Other Organisms
Research on TVP23 homologs in other organisms provides valuable insights into its potential functions in Y. lipolytica. The TVP23 protein family is conserved across species, from yeast to humans, suggesting fundamental roles in cellular processes . In mammals, TVP23B has been identified as a transmembrane protein that regulates host-microbe interactions through its effects on intestinal barrier function .
A recent study revealed that mammalian TVP23B controls the homeostasis of Paneth cells and the function of goblet cells, leading to the production of antimicrobial peptides and maintenance of the mucus layer in the intestine . TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis . The Golgi proteomes of cells deficient in these proteins show a common deficiency of several critical glycosylation enzymes, highlighting the importance of TVP23 family proteins in maintaining proper Golgi function .
While the specific functions of Y. lipolytica TVP23 may differ from its mammalian counterparts due to the unique cellular and physiological context, these findings suggest potential roles for TVP23 in maintaining Golgi structure and function, particularly in relation to glycosylation and vesicular trafficking. The conservation of these functions across evolutionarily distant species underscores the fundamental importance of TVP23 in cellular organization and function .
The identification of protein interaction partners for TVP23 homologs in other organisms provides a framework for investigating similar interactions in Y. lipolytica. Such studies could reveal the molecular mechanisms by which TVP23 contributes to vesicular trafficking and Golgi function in this biotechnologically important yeast species .
Biotechnological Implications
Understanding TVP23's role in the secretory pathway of Y. lipolytica has significant implications for biotechnological applications. Y. lipolytica has emerged as a promising platform for the production of recombinant proteins, lipids, and other valuable compounds, largely due to its exceptional secretory capabilities . Knowledge of the molecular mechanisms underlying these capabilities, including the function of proteins like TVP23, can inform strategies for optimizing Y. lipolytica for specific biotechnological applications.
Research on Y. lipolytica's secretory pathway has already identified various approaches for enhancing protein secretion, such as engineering secretion signals, expanding the ER, and co-expressing components of the secretory pathway . These strategies have resulted in substantial improvements in protein secretion yields, with combined approaches achieving up to 50-fold increases in some cases . Investigating TVP23's role in this context could reveal additional targets for engineering improved secretion efficiency.
The molecular genetic toolbox for Y. lipolytica continues to expand, with the development of isogenic strains, genome sequencing, and plasmids for targeted overexpression and fluorescent tagging . These resources enable more sophisticated investigations of proteins like TVP23, including live-cell imaging studies to visualize its dynamics and interactions within the Golgi apparatus. The "Yarrowia lipolytica Cell Atlas," a collection of strains with fluorescently tagged organelles, provides a valuable resource for studying the localization and function of proteins in relation to specific subcellular compartments .
As Y. lipolytica is increasingly utilized for applications in biofuels and natural products, insights into its cellular organization and protein trafficking processes become more valuable . The study of TVP23 and other components of the secretory pathway contributes to a more comprehensive understanding of Y. lipolytica's biology, facilitating its rational engineering for diverse biotechnological applications.
Exploring TVP23's Protein Interaction Network
Future research on Y. lipolytica TVP23 could focus on identifying and characterizing its interaction partners within the Golgi apparatus and broader secretory pathway. The finding that mammalian TVP23B interacts with YIPF6 suggests that Y. lipolytica TVP23 may engage in similar protein-protein interactions that are critical for Golgi function . Systematic approaches such as affinity purification coupled with mass spectrometry could be employed to identify TVP23's interaction network in Y. lipolytica.
Understanding these interactions would provide insights into the molecular mechanisms by which TVP23 contributes to vesicular trafficking and Golgi organization. For example, interactions with proteins involved in vesicle formation, targeting, or fusion would suggest specific roles for TVP23 in these processes. Similarly, interactions with glycosylation enzymes or other Golgi-resident proteins would indicate potential functions in protein modification or Golgi structure maintenance .
The elucidation of TVP23's interaction network could also reveal connections to other cellular processes, such as lipid metabolism or stress responses, that might be relevant to Y. lipolytica's biotechnological applications. For instance, if TVP23 interacts with proteins involved in lipid synthesis or trafficking, this could have implications for engineering Y. lipolytica strains with enhanced lipid production capabilities. Comprehensive characterization of these interactions would contribute to a more integrated understanding of TVP23's function in the broader context of cellular physiology.
Additionally, comparative studies of TVP23 interactions across different growth conditions or genetic backgrounds could reveal how this protein's function is regulated in response to environmental changes or metabolic demands. Such investigations would enhance our understanding of the dynamic nature of the secretory pathway and its adaptation to varying cellular requirements.
Engineering TVP23 for Enhanced Secretory Capacity
Based on our understanding of TVP23's role in vesicular trafficking, targeted modifications of this protein could potentially enhance Y. lipolytica's secretory capacity for biotechnological applications. Engineering approaches might include overexpression of native or modified TVP23, introduction of specific mutations to alter its function, or expression of TVP23 homologs from other organisms with potentially advantageous properties .
The development of Y. lipolytica strains with optimized TVP23 expression or function could lead to improvements in protein secretion efficiency, which is particularly valuable for the production of enzymes, biopharmaceuticals, or other proteins of interest . By enhancing the efficiency of vesicular trafficking within the Golgi apparatus, such modifications might alleviate bottlenecks in the secretory pathway that currently limit protein production yields.
Furthermore, engineering approaches targeting TVP23 could be combined with other strategies for enhancing protein secretion, such as optimization of secretion signals, expansion of the ER, or co-expression of chaperones or other secretory pathway components . These integrated approaches might achieve synergistic improvements in secretion efficiency, further enhancing Y. lipolytica's utility as a protein production platform.
Beyond protein secretion, modifications of TVP23 might also impact other aspects of Y. lipolytica's physiology relevant to biotechnological applications, such as lipid accumulation or stress tolerance. The complex interplay between vesicular trafficking, lipid metabolism, and cellular homeostasis suggests that engineering TVP23 could have multifaceted effects on Y. lipolytica's properties as a biotechnological platform .
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please specify them when placing your order. We will fulfill your request if possible.
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 of liquid forms is 6 months at -20°C/-80°C. Lyophilized forms typically have a 12-month shelf life at -20°C/-80°C.
Target Background
KEGG: yli:YALI0D25036g
STRING: 4952.XP_503257.1
Q&A
What is Yarrowia lipolytica Golgi apparatus membrane protein TVP23?
TVP23 is a conserved transmembrane protein found in the Golgi apparatus of Yarrowia lipolytica, a non-conventional yeast species. It consists of 180 amino acids and is involved in regulating vesicular trafficking to and from the Golgi compartments. The protein belongs to a conserved family represented across species from yeast to mammals and plants, indicating fundamental importance in eukaryotic cell biology. In recombinant form, it can be expressed with an N-terminal His tag in E. coli expression systems . Homologs of this protein in other organisms, such as TVP23B in mammals, have been implicated in crucial cellular functions including Golgi protein localization and trafficking .
What experimental approaches are most effective for studying TVP23 localization and dynamics?
For detailed investigation of TVP23 localization and dynamics, researchers should consider these methodological approaches:
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Fluorescence Microscopy Techniques:
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Confocal microscopy using fluorescent protein-tagged TVP23 (e.g., GFP-TVP23) to visualize precise localization within Golgi subcompartments
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Live-cell imaging with spinning disk confocal microscopy for real-time trafficking dynamics
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FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility within membranes
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Super-resolution microscopy (STED, PALM, STORM) for nanoscale localization studies
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Biochemical Approaches:
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Subcellular fractionation followed by Western blotting to confirm Golgi enrichment
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Protease protection assays to determine membrane topology
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Chemical crosslinking combined with mass spectrometry to identify proximal interaction partners
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Genetic Strategies:
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CRISPR/Cas9-mediated knockout followed by complementation with fluorescent protein-tagged variants
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Inducible expression systems to control TVP23 levels and observe acute effects
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By comparing results from mammalian TVP23B studies, which show specific binding with another Golgi protein YIPF6 , researchers can explore whether similar interactions occur with Yarrowia lipolytica TVP23, providing insights into conserved trafficking mechanisms.
How does TVP23 contribute to glycosylation processes in the Golgi apparatus?
Studies of the mammalian homolog TVP23B reveal a critical connection between TVP23 family proteins and glycosylation machinery:
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Glycosylation Enzyme Regulation:
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Research Approach for Y. lipolytica TVP23:
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Comparative proteomics analysis of wild-type versus TVP23-deficient Y. lipolytica Golgi preparations
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Glycomic profiling using mass spectrometry to identify specific glycan alterations
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Activity assays for key glycosyltransferases in the presence and absence of TVP23
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Trafficking studies of model glycoproteins through the secretory pathway
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Mechanistic Models:
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TVP23 may function as a scaffold protein that supports proper localization of glycosylation enzymes
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Alternatively, it might regulate vesicular transport between Golgi cisternae, affecting the sequential processing of glycoproteins
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TVP23 could also influence Golgi pH or ion concentrations, indirectly affecting glycosylation enzyme activity
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A systematic investigation using these approaches would elucidate whether the glycosylation-related functions observed in mammalian TVP23B are conserved in the yeast homolog, providing insights into fundamental mechanisms of Golgi glycosylation .
What methodological challenges exist in expressing and purifying functional recombinant TVP23?
As a multi-pass membrane protein, TVP23 presents several technical challenges for recombinant expression and purification:
Successful purification of functionally active TVP23 would provide valuable material for structural studies and in vitro functional assays, significantly advancing understanding of this protein family.
How can protein-protein interaction networks of TVP23 be mapped comprehensively?
Mapping the TVP23 interactome requires multiple complementary approaches:
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Proximity-Based Labeling Methods:
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BioID approach: Fusion of TVP23 with a biotin ligase (BirA*) to biotinylate proximal proteins
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APEX2 approach: TVP23-APEX2 fusion for peroxidase-catalyzed labeling of neighboring proteins
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These methods are particularly valuable for membrane proteins like TVP23 as they capture transient interactions in native cellular environments
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Affinity Purification-Mass Spectrometry:
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Co-immunoprecipitation of tagged TVP23 followed by mass spectrometry
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Crosslinking strategies to stabilize interactions before purification
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Quantitative comparison between specific pulldowns and controls
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Genetic Interaction Mapping:
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Synthetic genetic array analysis in yeast to identify genetic interactions
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CRISPR screens to identify genes with synergistic effects when mutated alongside TVP23
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Validation and Characterization:
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Co-localization studies of TVP23 with candidate interactors using multi-color fluorescence microscopy
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Bimolecular fluorescence complementation to confirm direct interactions in vivo
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In vitro binding assays with purified components to determine binding affinities
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Based on mammalian TVP23B studies, which identified YIPF6 as a key interactor affecting glycosylation enzyme composition in the Golgi , researchers should particularly focus on identifying YIPF family homologs in Y. lipolytica and characterizing their interactions with TVP23.
What are the optimal buffer conditions for maintaining TVP23 stability during purification?
Membrane proteins like TVP23 require carefully optimized buffer conditions to maintain stability throughout purification:
| Buffer Component | Recommended Range | Rationale | Optimization Strategy |
|---|---|---|---|
| pH | 7.0-7.5 | Mimics physiological Golgi pH | Screen pH in 0.2 unit increments |
| Salt (NaCl) | 150-300 mM | Shields electrostatic interactions | Test multiple concentrations for optimal stability |
| Glycerol | 5-10% | Prevents aggregation | Balance stability benefits against viscosity for chromatography |
| Detergent | 2-3x CMC | Maintains solubilization without excess micelles | Screen multiple detergent types (DDM, LMNG, LDAO) |
| Reducing Agent | 1-5 mM DTT or TCEP | Prevents disulfide formation | Include in all buffers throughout purification |
For the His-tagged recombinant TVP23 , additional considerations include:
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Low imidazole (10-20 mM) in wash buffers to reduce non-specific binding
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Elution with gradient to 250-300 mM imidazole
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Consider buffer exchange immediately after elution to remove imidazole, which can destabilize some proteins
Thermal stability assays (differential scanning fluorimetry) using various buffer compositions can identify optimal conditions that maximize protein stability. For long-term storage, flash-freezing aliquots in liquid nitrogen with 10% glycerol as cryoprotectant is recommended based on standard practices for membrane proteins.
How can researchers verify proper folding and functionality of recombinant TVP23?
Verifying the structural integrity and functionality of purified recombinant TVP23 requires multiple complementary approaches:
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Structural Integrity Assessment:
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Circular dichroism spectroscopy to evaluate secondary structure content
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Tryptophan fluorescence to assess tertiary structure
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Size exclusion chromatography to confirm homogeneity and absence of aggregation
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Limited proteolysis to probe for well-folded, protease-resistant domains
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Membrane Insertion Analysis:
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Liposome flotation assays to confirm membrane association
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Protease protection assays to verify proper topology
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Electron microscopy of TVP23-containing proteoliposomes
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Functional Validation:
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Comparative Analysis:
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Parallel characterization of wild-type and mutant variants
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Comparison with properties of homologs from other species
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Correlation of in vitro properties with in vivo functionality
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Since the recombinant His-tagged TVP23 has been successfully expressed in E. coli , researchers can use this as a starting point for extensive characterization using the approaches outlined above.
What experimental design would best elucidate the role of TVP23 in vesicular trafficking?
A comprehensive investigation of TVP23's role in vesicular trafficking should incorporate these experimental approaches:
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Loss-of-Function Studies:
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Generation of TVP23 knockout strains in Y. lipolytica
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Phenotypic characterization focusing on secretory pathway defects
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Electron microscopy to examine Golgi morphology changes
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Analysis of protein trafficking using model cargo proteins with different destinations
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Real-Time Trafficking Assays:
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Synchronized cargo release using temperature-sensitive mutants or retention systems
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Live-cell imaging with dual-color labeling of TVP23 and trafficking markers
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RUSH (Retention Using Selective Hooks) system adapted for yeast to monitor synchronized protein trafficking
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Biochemical Trafficking Assays:
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Vesicle immunoisolation using TVP23 antibodies
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In vitro reconstitution of vesicle budding with recombinant components
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Analysis of post-translational modifications as markers for trafficking progression
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Comparative Analysis with Known Trafficking Regulators:
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Double mutant analysis with genes involved in known trafficking pathways
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Localization studies under conditions that block specific trafficking steps
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Chemical genetics approach using trafficking inhibitors
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Proteomics Approaches:
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Analysis of vesicle composition in wild-type versus TVP23-deficient cells
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Cargo profiling to identify specifically affected transport pathways
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Temporal analysis of protein associations during trafficking events
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Research on mammalian TVP23B has revealed its importance for specific trafficking events related to intestinal cell function , suggesting Y. lipolytica TVP23 may similarly regulate specialized trafficking pathways that could be identified through these systematic approaches.
How do structural differences between TVP23 homologs relate to their specialized functions?
TVP23 homologs across species share core structural elements but display important variations that correlate with specialized functions:
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Conserved Structural Elements:
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Multi-pass transmembrane topology is preserved across homologs
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Key cytoplasmic domains likely involved in trafficking machinery interactions
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Golgi retention signals ensuring proper localization
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Divergent Functional Domains:
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The mammalian TVP23B contains specialized domains supporting interactions with YIPF6 and influencing Paneth and goblet cell function
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Plant homolog (Echidna) contains plant-specific motifs related to cell elongation functions
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Y. lipolytica TVP23's 180-amino acid sequence may contain yeast-specific elements
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Structure-Function Correlation Methodology:
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Multiple sequence alignment of TVP23 family proteins to identify conserved versus variable regions
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Homology modeling based on any available structural data
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Domain swapping experiments between homologs to identify function-specific regions
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Systematic mutagenesis of conserved versus variable domains
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Evolutionary Considerations:
Understanding these structural-functional relationships would provide insight into both the fundamental mechanisms of vesicular trafficking and the evolution of specialized trafficking pathways in different organisms.
What can researchers learn from TVP23B's role in mammalian cells to guide studies in Yarrowia lipolytica?
Research on mammalian TVP23B provides valuable direction for investigating Y. lipolytica TVP23:
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Key Insights from TVP23B Studies:
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Translatable Research Directions:
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Identify potential YIPF family homologs in Y. lipolytica and investigate interactions with TVP23
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Examine effects of TVP23 deletion on secretory products in Y. lipolytica
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Analyze glycosylation patterns of secreted proteins in TVP23-deficient yeast
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Investigate TVP23's role in maintaining Golgi proteome composition
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Methodological Transfer:
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Apply comparative proteomics approaches used in mammalian studies to yeast
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Adapt interaction mapping techniques to identify conserved binding partners
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Utilize similar imaging approaches to track protein localization and trafficking
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Experimental Design Example:
These translations from mammalian to yeast research will help establish whether TVP23's fundamental functions are conserved across evolution while identifying yeast-specific aspects of its biology.
How might TVP23 be engineered to improve recombinant protein production in Yarrowia lipolytica?
The potential for engineering TVP23 to enhance Y. lipolytica as an expression system is supported by several considerations:
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Enhancing Secretory Pathway Efficiency:
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Overexpression of TVP23 might increase vesicular trafficking capacity
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Co-expression with interacting partners (YIPF family proteins) could synergistically improve trafficking
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Chimeric TVP23 incorporating functional domains from mammalian homologs may enhance specific secretory functions
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Application to Existing Y. lipolytica Expression Systems:
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Y. lipolytica has already been demonstrated as an effective host for recombinant proteins, including virus-like particles (VLPs)
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TVP23 engineering could complement existing strategies using strong promoters (TEF1) and multicopy integration
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The integration of TVP23 modifications with optimized expression cassettes could further enhance productivity
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Targeted Improvements for Glycoprotein Production:
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Given TVP23B's role in glycosylation enzyme regulation , modified TVP23 could enhance specific glycosylation pathways
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Engineering efforts could focus on improving therapeutic glycoprotein production with more homogeneous glycoforms
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Knockout or modification of TVP23 might simplify glycosylation patterns for easier downstream processing
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Implementation Strategy:
This engineering approach would build upon Y. lipolytica's demonstrated potential as a platform for recombinant protein production, particularly for complex products like VLPs .
What emerging technologies could advance our understanding of TVP23 function?
Several cutting-edge technologies offer promising avenues for deeper investigation of TVP23:
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Cryo-Electron Tomography:
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Visualizing TVP23's native organization within the Golgi membrane at near-atomic resolution
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Mapping the 3D architecture of TVP23-containing trafficking intermediates
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Examining structural changes in Golgi morphology in TVP23-deficient cells
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In-Cell NMR Spectroscopy:
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Monitoring structural dynamics of TVP23 in living cells
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Detecting conformational changes during trafficking processes
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Identifying binding events with interacting proteins
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Optogenetic and Chemogenetic Control:
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Acute temporal control of TVP23 function using light-sensitive protein domains
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Rapid induction of protein-protein interactions to probe trafficking mechanisms
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Targeted protein degradation to achieve acute TVP23 depletion
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Spatial Transcriptomics and Proteomics:
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Mapping the spatial organization of mRNAs and proteins in TVP23-deficient cells
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Identifying compartment-specific changes in protein composition
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Correlating local molecular changes with alterations in organelle morphology
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Advanced AI-Driven Structure Prediction:
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Using AlphaFold or similar tools to predict TVP23 structure with high confidence
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Molecular dynamics simulations to model membrane interactions and protein dynamics
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In silico screening for small molecules that could modulate TVP23 function
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These emerging technologies, combined with traditional approaches, would provide unprecedented insights into the molecular mechanisms of TVP23 function in Golgi trafficking and organization.
