Recombinant Human Cell cycle control protein 50B (TMEM30B)

What is the basic structure and cellular localization of TMEM30B?

TMEM30B, also known as CDC50B, is a membrane protein with 2 transmembrane domains and an extracellular loop containing 3 cysteines and an N-glycosylation site. The protein consists of 351 amino acids in humans and is localized primarily to the endoplasmic reticulum (ER) . Topological studies have confirmed that both the N-terminus and C-terminus of TMEM30B face the cytoplasm, with the loop region extending into the lumen of the ER . The protein belongs to the CDC50/LEM3 family and is encoded by the TMEM30B gene located on chromosome 14 in humans .

Experimental approaches for investigating TMEM30B structure include:

• Immunofluorescence microscopy for localization studies

• Protease protection assays for topology determination

• Bioinformatic tools such as WoLF PSORT, PrediSi, and SignalP for prediction of subcellular localization and signal sequences

What are the primary functional roles of TMEM30B in cellular physiology?

TMEM30B serves as an accessory component of the P4-ATPase flippase complex, which is crucial for establishing and maintaining phospholipid asymmetry across cellular membranes . The protein plays several important physiological roles:

• Contributes to the maintenance of asymmetric distribution of phospholipids across membrane bilayers

• Facilitates the uptake of lipid signaling molecules

• Participates in vesicle formation

• Regulates the export of α subunits (ATP8B1, ATP8A1, ATP8B2, and ATP8B4) from the endoplasmic reticulum to the plasma membrane

These functions are essential for normal cellular processes including membrane integrity, signaling, and trafficking. The phospholipid flippase activity requires TMEM30B to partner with P4-ATPases, forming a functional complex that catalyzes the ATP-dependent translocation of phospholipids from the exoplasmic to the cytoplasmic leaflet of cellular membranes .

How does TMEM30B expression vary across different tissues?

TMEM30B exhibits a tissue-specific expression pattern with notable presence in:

• Pancreatic islets

• Kidney

• Prostate

• Various tumor tissues including lung carcinoid, parathyroid tumor, bladder tumor, meningioma, and pancreatic cancer

Expression profiling methods such as RNA-seq, quantitative PCR, and immunohistochemistry can be employed to detect tissue-specific TMEM30B expression. When designing such experiments, researchers should consider the multiple isoforms of TMEM30B that have been identified, as differential expression of specific isoforms may occur in different tissues or pathological states .

How does TMEM30B contribute to phospholipid translocation mechanisms?

TMEM30B functions as a critical beta-subunit of P4-ATPase flippase complexes that orchestrate phospholipid translocation across biological membranes . This process involves:

• Formation of a heterodimeric complex between TMEM30B and various P4-ATPase α-subunits

• ATP-dependent translocation of specific phospholipids from the exoplasmic to the cytoplasmic leaflet

• Generation and maintenance of phospholipid asymmetry critical for membrane integrity and function

Research methodologies to study TMEM30B-mediated phospholipid flipping include:

• Fluorescently labeled phospholipid translocation assays

• Reconstitution of purified protein complexes in liposomes

• CRISPR/Cas9-mediated gene editing to create knockout or knockdown models

• Co-immunoprecipitation studies to identify interacting P4-ATPase partners

The mechanism likely involves conformational changes in the protein complex that facilitate the movement of phospholipid headgroups through a hydrophilic pathway while keeping the hydrophobic tails within the membrane environment .

What role does TMEM30B play in the export of P4-ATPases from the ER to plasma membrane?

TMEM30B serves as a crucial chaperone that regulates the export of P4-ATPase α-subunits (ATP8B1, ATP8A1, ATP8B2, and ATP8B4) from the endoplasmic reticulum to the plasma membrane . This function is essential for:

• Proper trafficking and localization of P4-ATPases

• Assembly of functional flippase complexes at appropriate cellular membranes

• Maintenance of membrane homeostasis and lipid asymmetry

The process likely involves:

• Recognition and binding of newly synthesized P4-ATPase α-subunits in the ER

• Facilitation of proper folding and quality control

• Escort through the secretory pathway to final destinations

Experimental approaches to investigate this process include:

• Pulse-chase experiments tracking the movement of tagged P4-ATPases

• Confocal microscopy with fluorescently labeled proteins

• Brefeldin A treatment to block ER-to-Golgi transport

• Co-localization studies with markers for different compartments of the secretory pathway

How do the different isoforms of TMEM30B affect its functional properties?

Multiple transcript variants of TMEM30B have been identified, suggesting potential functional diversity . In zebrafish, for example, four different mRNA transcripts (tmem30b-201, tmem30b-202, tmem30b-203, and tmem30b-204) have been annotated with lengths ranging from 749 to 3,600 nucleotides . In humans, at least three isoforms have been detected (ENST00000555868.1, ENST00000554497.1, and ENST00000557163.1) .

Research approaches to study isoform-specific functions include:

• Isoform-specific qPCR to quantify expression levels

• Cloning and overexpression of individual isoforms

• Isoform-specific knockdown using siRNA or CRISPR technologies

• Comparative functional assays of different isoforms

The functional differences between these isoforms remain largely uncharacterized, presenting an important avenue for future research. Distinct isoforms may exhibit tissue-specific expression patterns, differential binding affinities for P4-ATPase partners, or varied regulatory properties in phospholipid translocation .

What is the relationship between TMEM30B expression and cancer progression?

TMEM30B expression has been implicated in several cancer types, with evidence suggesting both downregulation and potential prognostic significance:

Methodological approaches for investigating TMEM30B in cancer include:

• Tissue microarray analysis for expression profiling

• Kaplan-Meier survival analysis correlating expression with clinical outcomes

• In vitro studies examining effects of TMEM30B knockdown or overexpression on cancer cell phenotypes

• Analysis of mutation databases and cancer genomics datasets

What genetic alterations of TMEM30B have been identified in cancer?

Several genetic alterations affecting TMEM30B have been observed in cancer tissues:

• Mutations:

  • Potentially damaging mutations in TMEM30B have been identified in clear cell Renal Cell Carcinoma (ccRCC)

  • Specific mutation hotspots or functional consequences require further characterization

• Chromosomal Aberrations:

  • Deletions in the TMEM30B locus have been found in nearly 30% of ccRCC tumors

  • These deletions may contribute to the downregulation of TMEM30B expression observed in cancer

Experimental approaches to study TMEM30B genetic alterations include:

• Next-generation sequencing of tumor samples

• Copy number variation analysis

• Functional characterization of identified mutations using site-directed mutagenesis

• CRISPR/Cas9-mediated introduction of cancer-associated mutations in cell models

The relatively high frequency of TMEM30B locus deletions in ccRCC (30%) suggests a potential tumor suppressor role, though additional functional studies are needed to confirm this hypothesis .

How might alterations in TMEM30B contribute to disease pathogenesis?

Alterations in TMEM30B function may contribute to disease pathogenesis through several mechanisms:

• Disruption of membrane phospholipid asymmetry:

  • Altered phospholipid distribution can affect membrane integrity and cellular functions

  • May impact cell signaling, apoptosis, and interactions with the extracellular environment

• Impaired trafficking of P4-ATPases:

  • Defective export of P4-ATPases from ER to plasma membrane

  • Mislocalization of flippase activity within cellular compartments

• Downstream effects on cellular processes:

  • Changes in vesicle formation and membrane trafficking

  • Alterations in lipid signaling pathways

  • Potential impact on cell cycle regulation (suggested by its name as Cell cycle control protein 50B)

Methodological approaches for investigating these mechanisms include:

• Analysis of phospholipid distribution using fluorescent lipid analogs

• Examination of P4-ATPase localization in cells with altered TMEM30B expression

• Characterization of membrane properties such as fluidity and permeability

• Assessment of downstream signaling pathway activation

The association of TMEM30B alterations with cancer progression suggests that disruption of normal phospholipid asymmetry and membrane homeostasis may contribute to malignant transformation or tumor progression .

What are the optimal approaches for studying TMEM30B localization and topology?

For comprehensive characterization of TMEM30B localization and topology, researchers should consider a multi-method approach:

• Subcellular Localization Studies:

  • Immunofluorescence microscopy with co-localization markers for specific organelles

  • Subcellular fractionation followed by Western blotting

  • Live-cell imaging with fluorescently tagged TMEM30B

• Membrane Topology Determination:

  • Protease protection assays to identify cytoplasmic versus lumenal domains

  • Glycosylation mapping to identify lumenal domains

  • Cysteine accessibility methods using membrane-permeable and impermeable sulfhydryl reagents

  • Epitope insertion combined with antibody accessibility testing

• Computational Prediction Methods:

  • WoLF PSORT for subcellular localization prediction

  • PrediSi, SignalBlast, PolyPhobius for signal sequence prediction

  • TargetP and SignalP for targeting sequence analysis

Previous experimental verification has confirmed that TMEM30B is localized to the endoplasmic reticulum with both N-terminus and C-terminus facing the cytoplasm . This topology is consistent with its role as a partner for P4-ATPases, facilitating their export from the ER to the plasma membrane.

What are the best expression systems and purification methods for recombinant TMEM30B production?

Production of functional recombinant TMEM30B requires careful consideration of expression systems and purification strategies:

• Expression Systems:

  • Mammalian expression (HEK293, CHO cells) for proper post-translational modifications

  • Insect cell systems (Sf9, High Five) for higher yields while maintaining eukaryotic processing

  • Yeast systems (P. pastoris, S. cerevisiae) as alternatives with eukaryotic processing machinery

  • E. coli systems may be challenging due to the transmembrane nature but can be used with proper fusion tags

• Expression Strategies:

  • Fusion tags: His6, FLAG, GST, or MBP to facilitate purification

  • Codon optimization for the chosen expression system

  • Inducible promoters for controlled expression

  • Co-expression with P4-ATPase partners may improve stability

• Purification Methods:

  • Detergent solubilization (DDM, LMNG, or other mild detergents)

  • Affinity chromatography based on fusion tags

  • Size exclusion chromatography for final polishing

  • Consideration of lipid addition during purification to maintain stability

• Functional Validation:

  • Circular dichroism to assess secondary structure

  • Binding assays with P4-ATPase partners

  • Reconstitution into liposomes for functional studies

When designing recombinant TMEM30B constructs, researchers should consider the inclusion of the complete coding sequence (351 amino acids) and potentially incorporate flexible linkers if fusion tags are used .

What methodological approaches are most effective for studying TMEM30B interactions with P4-ATPases?

Investigating the interactions between TMEM30B and P4-ATPases requires specialized techniques:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to identify native complexes

  • Proximity labeling techniques (BioID, APEX) to map interaction networks

  • FRET or BiFC to visualize interactions in living cells

  • Yeast two-hybrid or mammalian two-hybrid screens to identify interaction domains

• Structural Studies:

  • Cryo-electron microscopy of purified complexes

  • X-ray crystallography (challenging but potentially informative)

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cross-linking mass spectrometry to identify proximal residues

• Functional Assays:

  • Phospholipid flipping assays in reconstituted systems

  • Mutagenesis of potential interaction sites followed by binding and activity assays

  • Trafficking assays to monitor P4-ATPase export from ER in presence/absence of TMEM30B

• Computational Approaches:

  • Molecular docking simulations

  • Sequence co-evolution analysis to predict interaction interfaces

  • Structural modeling based on related protein complexes

These methods can help elucidate the specific mechanisms by which TMEM30B interacts with its P4-ATPase partners (ATP8B1, ATP8A1, ATP8B2, and ATP8B4) and how these interactions facilitate phospholipid flipping and protein trafficking .

How does TMEM30B contribute to the post-transcriptional regulation of gene expression?

Emerging evidence suggests potential roles for TMEM30B in post-transcriptional regulation mechanisms:

• Interaction with RNA-Binding Proteins:

  • TMEM30B may interact with splicing regulators or other RNA-binding proteins

  • Could potentially influence pre-mRNA processing or stability

• Connection to BRG1 and FIRΔexon2:

  • Research has identified a potential relationship between TMEM30B, BRG1 (an ATPase subunit of the SWI/SNF chromatin remodeling complex), and FIRΔexon2 (a splicing variant of the far-upstream element-binding protein interacting repressor)

  • FIRΔexon2 may acetylate H3K27 on the BRG1 promoter and suppress BRG1 expression post-transcriptionally

  • BRG1 in turn suppresses Snai1, a transcriptional suppressor of E-cadherin that prevents cancer invasion and metastasis

• Potential Impact on RNA Splicing:

  • TMEM30B alterations might influence alternative splicing patterns

  • Research has shown that siRNA targeting related proteins can alter pre-mRNA splicing of genes like FGF8

These complex regulatory networks require sophisticated experimental approaches:

• RNA immunoprecipitation to identify associated transcripts

• CLIP-seq to map RNA-protein interaction sites

• Transcriptome analysis following TMEM30B modulation

• Splicing reporter assays to assess effects on specific splicing events

The emerging connections between TMEM30B and post-transcriptional regulation open new research directions beyond its established role in phospholipid translocation .

What is the relationship between TMEM30B and other CDC50 family members in cellular function?

TMEM30B belongs to the CDC50/LEM3 family, which includes other members with related functions:

• Functional Overlap and Specialization:

  • TMEM30A (CDC50A) is the most extensively studied member

  • Different CDC50 family members may associate with distinct subsets of P4-ATPases

  • Tissue-specific expression patterns suggest specialized roles

• Compensatory Mechanisms:

  • Potential redundancy between family members

  • Altered expression of other CDC50 proteins in response to TMEM30B deficiency

  • Differential regulation in disease states

• Evolutionary Conservation:

  • CDC50 proteins are highly conserved across species

  • Zebrafish tmem30b shows significant homology to human TMEM30B

  • Functional studies in model organisms can provide valuable insights

Research approaches to investigate these relationships include:

• Comparative expression analysis of CDC50 family members across tissues

• Co-immunoprecipitation studies to identify specific P4-ATPase partners

• Knockout/knockdown studies with single and multiple family members

• Complementation experiments to test functional redundancy

Understanding the specific roles of TMEM30B within the broader CDC50 family context may provide insights into tissue-specific functions and disease mechanisms .

How might TMEM30B be exploited as a therapeutic target or biomarker in disease?

The involvement of TMEM30B in cancer and other diseases suggests potential applications as a therapeutic target or biomarker:

The relationship between TMEM30B expression and clinical outcomes in multiple cancer types suggests significant potential for translational applications, though additional validation studies are needed before clinical implementation .