GO:1990531 phospholipid-translocating ATPase complex: Components, Assembly and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:1990531 describes the phospholipid-translocating ATPase complex, a protein assembly that functions as a phospholipid-translocating P-Type ATPase.
• The complex is also known as the P4-ATPase complex, flippase complex, or APLT complex, and includes yeast complexes such as Lem3p-Dnf1p.
• In yeast, the Lem3p-Dnf1p complex is a well-characterized example, where Lem3p regulates Dnf1p independently of its phospholipid-translocating activity.
• The carboxyl-terminal region of Dnf1p plays a regulatory role in the complex, separate from the catalytic translocation function.
• Dysregulation of phospholipid-translocating ATPase complexes has been implicated in human diseases, including cystic fibrosis, where circular RNA dysregulation and cellular senescence were observed.
• Studying this complex requires combining genetic, biochemical, and imaging approaches, with CRISPR-based models offering precise tools for functional dissection.
Description
The phospholipid-translocating ATPase complex (GO:1990531) is a cellular component defined as a protein complex that functions as a phospholipid-translocating P-Type ATPase. This complex is responsible for the ATP-dependent movement of phospholipids across membrane bilayers, a process critical for maintaining membrane asymmetry and lipid homeostasis. In yeast, the Lem3p-Dnf1p complex serves as a prototypical example, where Dnf1p is the catalytic subunit and Lem3p is a regulatory subunit. Mutational analysis has revealed that Lem3p and the carboxyl-terminal region of Dnf1p have regulatory roles independent of the phospholipid-translocating activity of Dnf1p. This suggests that the complex has functions beyond simple lipid flipping, possibly in signaling or protein trafficking. Understanding GO:1990531 is therefore essential for researchers studying membrane biology, lipid asymmetry, and related diseases. Recent studies have also linked dysregulated circular RNAs and cellular senescence to cystic fibrosis, highlighting the broader relevance of phospholipid-translocating ATPase complexes in human pathology.
phospholipid-translocating ATPase complex At A Glance
| GO ID | GO:1990531 |
|---|---|
| GO term | phospholipid-translocating ATPase complex |
| Ontology | cellular_component |
| Synonym | P4-ATPase complex, flippase complex, APLT complex, Lem3p-Dnf1p complex |
| Major function | ATP-dependent translocation of phospholipids across membranes |
| Definition | A protein complex that functions as a phospholipid-translocating P-Type ATPase |
| Example complex | Lem3p-Dnf1p in yeast |
| Regulatory subunit | Lem3p regulates Dnf1p independently of its translocase activity |
What Is GO:1990531?
GO:1990531, the phospholipid-translocating ATPase complex, is a protein complex that acts as a phospholipid-translocating P-Type ATPase. This means it uses ATP hydrolysis to move phospholipids between the two leaflets of a membrane bilayer, typically flipping them from the exoplasmic to the cytoplasmic face. The complex is also referred to as the P4-ATPase complex, flippase complex, or aminophospholipid translocase complex. In yeast, a well-studied example is the Lem3p-Dnf1p complex, where Dnf1p is the catalytic subunit and Lem3p is an accessory subunit that regulates Dnf1p function.
Why Is phospholipid-translocating ATPase complex Important in Cell Biology?
The phospholipid-translocating ATPase complex is crucial for maintaining membrane lipid asymmetry, which affects cell signaling, vesicle trafficking, and apoptosis. Dysfunction of this complex can lead to defects in membrane organization and has been linked to diseases such as cystic fibrosis, where cellular senescence and circular RNA dysregulation are observed. Studying this complex provides insights into fundamental membrane biology and potential therapeutic targets.
• Maintains phospholipid asymmetry in biological membranes.
• Regulates membrane curvature and vesicle trafficking.
• Influences cell signaling pathways through lipid distribution.
• Its dysfunction is implicated in cystic fibrosis pathology.
• Serves as a model for P4-ATPase function and regulation.
• Lem3p and Dnf1p have regulatory roles independent of lipid translocation.
• Potential target for therapies modulating membrane lipid composition.
• Involved in cellular senescence and circular RNA dysregulation in disease.
What Happens During phospholipid-translocating ATPase complex?
Substrate Recognition and Binding
In simple terms: The complex first grabs a phospholipid molecule from one side of the membrane.
The phospholipid-translocating ATPase complex recognizes specific phospholipids, such as phosphatidylserine and phosphatidylethanolamine, on the exoplasmic leaflet of the membrane. In the yeast Lem3p-Dnf1p complex, Dnf1p is the catalytic subunit that binds the phospholipid substrate, while Lem3p assists in substrate recognition or presentation. Mutations in Lem3p can affect this binding step without abolishing ATPase activity, indicating a regulatory role.
ATP Hydrolysis and Conformational Change
In simple terms: The complex uses energy from ATP to change its shape and flip the lipid.
Upon substrate binding, the complex hydrolyzes ATP, which drives a conformational change in the catalytic subunit. This change moves the phospholipid across the membrane bilayer. The carboxyl-terminal region of Dnf1p is important for this process, as mutations there can uncouple ATP hydrolysis from lipid translocation.
Phospholipid Translocation
In simple terms: The lipid is flipped from one side of the membrane to the other.
The energy from ATP hydrolysis is used to translocate the phospholipid from the exoplasmic to the cytoplasmic leaflet. This flipping action is essential for maintaining membrane asymmetry. The Lem3p-Dnf1p complex specifically translocates aminophospholipids, and its activity is regulated by Lem3p.
Regulation and Recycling
In simple terms: The complex can be turned on or off and recycled within the cell.
The activity of the phospholipid-translocating ATPase complex is regulated by accessory subunits like Lem3p, which can modulate Dnf1p function independently of its translocase activity. Additionally, the carboxyl-terminal region of Dnf1p plays a regulatory role, possibly in protein stability or localization. Dysregulation of these regulatory mechanisms can lead to disease, as seen in cystic fibrosis where circular RNAs and senescence are altered.
Key Genes Involved in GO:1990531 phospholipid-translocating ATPase complex
The following genes and proteins are key components or regulators of the phospholipid-translocating ATPase complex, based on experimental evidence from yeast and human studies.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DNF1 | Catalytic subunit of the Lem3p-Dnf1p complex; phospholipid translocase | Mutations affect lipid flipping and regulation |
| LEM3 | Regulatory subunit; modulates Dnf1p activity | Regulates Dnf1p independently of translocase activity |
| DNF2 | Paralog of Dnf1; forms complex with Lem3p | Potential redundancy in lipid translocation |
| DRS2 | P4-ATPase; forms complex with Cdc50p | Involved in membrane trafficking |
| CDC50 | Accessory subunit for Drs2p | Required for Drs2p function |
| CRF1 | Accessory subunit for Dnf3p | Regulates Dnf3p complex |
| DNF3 | P4-ATPase; forms complex with Crf1p | Phospholipid translocation |
| ATP8A1 | Human P4-ATPase; flippase | Potential role in membrane asymmetry |
| ATP8A2 | Human P4-ATPase; flippase | Neurological function |
| ATP8B1 | Human P4-ATPase; flippase | Cholestasis and hearing loss |
| ATP8B2 | Human P4-ATPase; flippase | Membrane lipid regulation |
| ATP8B3 | Human P4-ATPase; flippase | Sperm function |
| ATP8B4 | Human P4-ATPase; flippase | Immune function |
| ATP9A | Human P4-ATPase; flippase | Endosomal trafficking |
| ATP9B | Human P4-ATPase; flippase | Golgi function |
| ATP10A | Human P4-ATPase; flippase | Lipid metabolism |
| ATP10B | Human P4-ATPase; flippase | Parkinson's disease risk |
| ATP10D | Human P4-ATPase; flippase | Glucose metabolism |
| ATP11A | Human P4-ATPase; flippase | Apoptosis and cancer |
| ATP11B | Human P4-ATPase; flippase | Membrane dynamics |
| ATP11C | Human P4-ATPase; flippase | B cell development |
How Is phospholipid-translocating ATPase complex Regulated?
The phospholipid-translocating ATPase complex is regulated by accessory subunits such as Lem3p, which modulates the catalytic subunit Dnf1p independently of its phospholipid-translocating activity. The carboxyl-terminal region of Dnf1p also plays a regulatory role, as mutations in this region affect complex function without altering ATPase activity. Additionally, dysregulated circular RNAs and cellular senescence have been implicated in cystic fibrosis, suggesting that broader cellular stress pathways may influence complex regulation.
phospholipid-translocating ATPase complex and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ATP8A2 | Neurological disorders | Knockout mouse or patient-derived iPSCs |
| ATP8B1 | Cholestasis and hearing loss | Liver-specific KO or knock-in models |
| ATP11A | Cancer and apoptosis | Cancer cell lines with overexpression or KO |
| LEM3 | Fungal pathogenesis | Yeast knockout and point mutation |
| DNF1 | Membrane trafficking defects | Yeast knockout and tagged knock-in |
Cystic Fibrosis
Dysregulated circular RNAs and cellular senescence have been observed in whole blood transcriptomes from cystic fibrosis patients, implicating a role for senescence in the disease. While the direct link to phospholipid-translocating ATPase complexes is not fully established, altered membrane lipid asymmetry could contribute to senescence and disease pathology.
Neurological Disorders
Human P4-ATPases such as ATP8A2 are critical for neurological function, and mutations in these genes can cause severe neurological disorders. The phospholipid-translocating ATPase complex is essential for maintaining neuronal membrane asymmetry, and its dysfunction may lead to neurodegeneration.
Cancer
Altered phospholipid asymmetry is a hallmark of cancer cells, and P4-ATPases like ATP11A are involved in apoptosis and cancer progression. The phospholipid-translocating ATPase complex may therefore be a potential therapeutic target in oncology.
From phospholipid-translocating ATPase complex-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the catalytic mechanism of Dnf1p? | Point mutation of catalytic residues in yeast |
| How does Lem3p regulate Dnf1p? | Knockout of LEM3 in yeast |
| What is the role of the carboxyl-terminus of Dnf1p? | Truncation or point mutations in yeast |
| How does the complex affect membrane asymmetry? | Tagged knock-in of Dnf1p for imaging |
| Can overexpression rescue lipid asymmetry? | Overexpression of DNF1 and LEM3 in yeast |
| What are the human disease implications? | Knockout of ATP8A2 in human cell lines |
How to Study the phospholipid-translocating ATPase complex Process
| Method | What It Measures | Typical Application |
|---|---|---|
| ATPase activity assay | ATP hydrolysis rate | Measure catalytic activity of Dnf1p |
| Lipid translocation assay | Phospholipid flipping | Assess flippase function |
| Fluorescence microscopy | Protein localization | Visualize complex assembly |
| RNA-seq | Gene expression changes | Identify dysregulated pathways |
| Circular RNA profiling | circRNA abundance | Link to cystic fibrosis |
| Senescence assays | Cellular senescence | Assess disease pathology |
| Yeast genetics | Gene function | Knockout and mutation studies |
| CRISPR-Cas9 | Precise gene editing | Create KO, KI, point mutations |
Genetic Knockout and Mutagenesis
Knockout of genes encoding subunits such as DNF1 or LEM3 in yeast can reveal their roles in phospholipid translocation and regulation. Point mutations in the catalytic domain or carboxyl-terminal region of Dnf1p can dissect functional domains.
Biochemical Assays
ATPase activity assays and phospholipid translocation assays using fluorescent lipids can measure the complex's function directly. These assays help determine if mutations affect catalysis or regulation.
Imaging and Localization
Fluorescence microscopy with tagged subunits (e.g., GFP-Dnf1p) can visualize complex localization and membrane dynamics. This is useful for studying assembly and trafficking.
Transcriptomics and Senescence Markers
RNA-seq and circular RNA profiling in patient samples, such as cystic fibrosis blood, can identify dysregulated pathways linked to the complex. Senescence markers can be used to assess cellular stress.
How CRISPR Can Be Used to Study GO:1990531 phospholipid-translocating ATPase complex
Knockout
CRISPR knockout of DNF1 or LEM3 in yeast or human orthologs can abolish complex function, revealing its role in membrane asymmetry and disease. Knockout models are essential for loss-of-function studies.
Point Mutation
CRISPR point mutations can introduce specific amino acid changes in catalytic or regulatory domains of Dnf1p, allowing precise dissection of function. For example, mutations in the carboxyl-terminal region can uncouple ATPase activity from lipid translocation.
Knock-in
CRISPR knock-in of tagged versions of Dnf1p or Lem3p enables live-cell imaging and proteomic studies. This helps track complex localization and interactions.
Overexpression
CRISPR activation or overexpression constructs can increase levels of complex subunits to study gain-of-function effects or rescue phenotypes. Overexpression of DNF1 and LEM3 can enhance lipid translocation.
How EDITGENE Supports phospholipid-translocating ATPase complex Research
Researchers studying phospholipid-translocating ATPase complex-related genes often need to determine whether a candidate gene is causally involved in membrane lipid asymmetry, vesicle trafficking, or disease. EDITGENE provides a comprehensive suite of CRISPR services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for phospholipid-translocating ATPase complex research.
Frequently Asked Questions About phospholipid-translocating ATPase complex
What is GO:1990531?
GO:1990531 is the phospholipid-translocating ATPase complex, a protein complex that functions as a phospholipid-translocating P-Type ATPase.
What genes are involved in phospholipid-translocating ATPase complex?
Key genes include DNF1, LEM3, DNF2, DRS2, CDC50, and human P4-ATPases such as ATP8A2 and ATP11A.
What is the function of phospholipid-translocating ATPase complex?
It uses ATP to flip phospholipids across membranes, maintaining lipid asymmetry and influencing signaling and trafficking.
How is phospholipid-translocating ATPase complex regulated?
It is regulated by accessory subunits like Lem3p and the carboxyl-terminal region of Dnf1p.
What diseases are associated with phospholipid-translocating ATPase complex?
Dysregulation has been linked to cystic fibrosis, neurological disorders, and cancer.
What is the Lem3p-Dnf1p complex?
It is a yeast phospholipid-translocating ATPase complex where Dnf1p is catalytic and Lem3p is regulatory.
How can I study phospholipid-translocating ATPase complex using CRISPR?
CRISPR knockout, point mutation, knock-in, and overexpression models can dissect gene function.
What methods are used to study phospholipid-translocating ATPase complex?
ATPase assays, lipid translocation assays, fluorescence microscopy, RNA-seq, and circular RNA profiling.
Is phospholipid-translocating ATPase complex involved in cancer?
Yes, altered phospholipid asymmetry is a hallmark of cancer, and P4-ATPases like ATP11A are implicated.
What is the role of cellular senescence in cystic fibrosis?
Dysregulated circular RNAs and senescence have been observed in cystic fibrosis patients, suggesting a role for senescence in disease.
Conclusion
The phospholipid-translocating ATPase complex (GO:1990531) is a critical cellular component that maintains membrane lipid asymmetry through ATP-dependent phospholipid translocation. Its regulatory subunits and carboxyl-terminal regions play key roles independent of catalytic activity. Dysregulation of this complex has been linked to cystic fibrosis, neurological disorders, and cancer, making it a promising research target. Advances in CRISPR-based models and bioinformatics will continue to unravel its mechanisms and therapeutic potential.
References
- 1. Noji T et al.. 2006. Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast.. Biochem Biophys Res Commun 344(1):323-31 PMID: 16600184
- 2. Salinas EA et al.. 2023. Discovery of dysregulated circular RNAs in whole blood transcriptomes from cystic fibrosis patients - implication of a role for cellular senescence in cystic fibrosis.. J Cyst Fibros 22(4):683-693 PMID: 37142522
