Recombinant Human Transmembrane protein 170A (TMEM170A)

What is TMEM170A and where is it localized in the cell?

TMEM170A is a human transmembrane protein that is conserved across major eukaryotic phyla but was functionally uncharacterized until recently. Research using transiently transfected TMEM170A-GFP, FLAG-TMEM170A, or myc-TMEM170A has consistently shown that the protein localizes specifically to both peripheral endoplasmic reticulum (ER) and nuclear envelope membranes . This localization has been confirmed through co-localization studies with established ER marker proteins such as calnexin and RTN4 . The specific localization pattern suggests TMEM170A plays a role in the organization and function of these membrane systems.

What is the molecular structure and size of TMEM170A?

TMEM170A is a small protein with a molecular weight of 15.25 kDa. Structural analysis reveals that it contains three transmembrane domains . According to predictions using TMPRED software, the N-terminus of TMEM170A is likely situated in the ER lumen, while the C-terminus extends into the cytoplasm . This membrane topology was experimentally verified using selective permeabilization experiments with digitonin (which permeabilizes only the plasma membrane) versus digitonin plus Triton X-100 (which permeabilizes all cellular membranes). In TMEM170A-GFP expressing cells, the GFP tag was accessible to anti-GFP antibodies after digitonin permeabilization, confirming the cytoplasmic orientation of the C-terminus .

How does TMEM170A affect endoplasmic reticulum morphology?

TMEM170A plays a critical role in determining ER morphology, specifically in regulating the balance between tubular and sheet structures within the ER network. Experimental evidence demonstrates that:

• Downregulation (silencing) of TMEM170A induces the formation of tubular ER, as observed through transmission electron microscopy (TEM) and 3D electron tomography . The silencing leads to the appearance of unorganized tubular ER aggregates which occasionally show some degree of tubule-tubule fusion but rarely form complete cisternal stacks.

• Conversely, overexpression of TMEM170A induces proliferation of ER sheets. High-resolution imaging by TEM and 3D electron tomography of cells overexpressing FLAG-TMEM170A reveals highly proliferated ER composed of extensive, well-organized sheet stacks decorated with membrane-bound ribosomes .

These findings indicate that TMEM170A is an ER-sheet-promoting protein, with its cellular concentration directly influencing the ratio between tubular ER and ER sheets .

What is the relationship between TMEM170A and nuclear envelope formation?

TMEM170A plays a significant role in nuclear envelope structure and formation. Research has shown that:

• Downregulation of TMEM170A causes nuclear envelope invaginations or evaginations, resulting in altered nuclear shape .

• TMEM170A silencing leads to an increase in nuclear surface area (by approximately 131% compared to control cells) and nuclear volume (by approximately 137%) .

• Conversely, overexpression of FLAG-TMEM170A causes a reduction in nuclear surface area (to about 84% of control cells) .

• TMEM170A silencing also affects inner nuclear membrane (INM) protein distribution, with reduced nuclear rim staining of proteins like LAP2β and emerin, and prominent mislocalization of LBR to the ER (in over 80% of silenced cells) .

These observations suggest that TMEM170A is crucial for maintaining proper nuclear envelope structure and the correct localization of INM proteins.

How does TMEM170A interact with reticulon family proteins like RTN4?

TMEM170A interacts with RTN4, a member of the reticulon family that is known to promote ER tubule formation. The relationship between these two proteins has been characterized as antagonistic:

• When both TMEM170A and RTN4 are simultaneously co-silenced, the phenotypes observed with singular TMEM170A silencing (related to ER structure, nuclear pore complex density, and nuclear envelope formation) are rescued .

• This rescue effect implies that TMEM170A and RTN4 have opposing functions in ER membrane organization, with RTN4 promoting tubular ER and TMEM170A promoting ER sheets .

• The antagonistic relationship also extends to effects on nuclear envelope formation and nuclear pore complex (NPC) formation .

This interaction provides insight into the molecular mechanisms that regulate the balance between different ER morphologies and indicates a coordinated system of proteins that work in opposition to maintain appropriate ER structure.

What experimental approaches can be used to study TMEM170A's role in ER sheet formation?

To investigate TMEM170A's role in ER sheet formation, researchers can employ multiple complementary approaches:

• Gene Silencing and Overexpression:

  • RNA interference techniques using specific siRNAs targeting TMEM170A to downregulate its expression .

  • Transient or stable transfection of TMEM170A constructs (such as FLAG-TMEM170A) for overexpression studies .

  • Creating stable cell lines expressing TMEM170A fused with tags like GFP for localization and functional studies .

• High-Resolution Imaging Techniques:

  • Transmission Electron Microscopy (TEM) to visualize ER ultrastructure in detail .

  • 3D electron tomography to create reconstructions of ER morphology and examine the organization of ER sheets and tubules .

  • Confocal microscopy with ER markers such as calnexin, CLIMP-63 (for ER sheets), and RTN4 (for ER tubules) to assess changes in ER structure in response to TMEM170A manipulation .

• Co-localization and Interaction Studies:

  • Immunofluorescence microscopy to examine co-localization of TMEM170A with other ER proteins .

  • Co-immunoprecipitation assays to detect physical interactions between TMEM170A and other ER-shaping proteins like RTN4 .

  • Proximity ligation assays to verify protein-protein interactions in situ.

• Membrane Topology Analysis:

  • Selective membrane permeabilization experiments using digitonin versus digitonin plus Triton X-100 to determine the orientation of protein domains relative to the membrane .

  • Protease protection assays to identify which regions of TMEM170A are accessible from different sides of the membrane.

• Quantitative Analysis of ER Morphology:

  • Morphometric analysis of ER sheets versus tubules in microscopy images .

  • Measuring ER sheet surface area and tubule length in control versus experimental conditions.

These methodologies, especially when combined, provide comprehensive insights into how TMEM170A influences ER morphogenesis and sheet formation.

How can one differentiate between the effects of TMEM170A and RTN4 in ER morphogenesis studies?

Differentiating between the effects of TMEM170A and RTN4 in ER morphogenesis requires experimental designs that can isolate and compare their individual and combined impacts:

• Sequential and Combinatorial Silencing/Overexpression:

  • Single silencing of TMEM170A or RTN4 to observe their individual effects on ER morphology .

  • Co-silencing of both proteins to assess potential rescue effects or synergistic changes .

  • Silencing one protein while overexpressing the other to examine dominant effects.

• Morphological Marker Analysis:

  • Utilize specific markers for different ER structures: CLIMP-63 for ER sheets and RTN4 itself or other reticulons for tubular ER .

  • Quantify the relative abundance and distribution of these markers under different experimental conditions.

• Rescue Experiments:

  • After silencing both proteins, reintroduce wild-type or mutant versions of each protein individually to determine which phenotypic aspects are rescued .

  • Use domain-specific mutants to identify which regions of each protein are essential for their opposing functions.

• Quantitative Ultrastructural Analysis:

  • Employ TEM and 3D electron tomography to measure specific parameters such as:

    • ER sheet length, width, and stacking

    • Tubule diameter, length, and branching frequency

    • Ribosome decoration patterns on ER membranes

  • Compare these measurements across different experimental conditions to detect subtle differences in how TMEM170A and RTN4 influence ER structure .

Through these approaches, researchers can systematically separate and characterize the specific contributions of TMEM170A and RTN4 to ER morphogenesis, despite their antagonistic relationship.

What are the methodological considerations when overexpressing or silencing TMEM170A in cellular models?

When manipulating TMEM170A expression levels in cellular models, researchers should consider several methodological factors to ensure robust and interpretable results:

• Expression System Selection:

  • For overexpression, consider using inducible systems (e.g., Tet-On/Off) to control expression levels and timing, preventing potential toxicity from constitutive high expression .

  • Choose appropriate tags (GFP, FLAG, myc) that don't interfere with protein function or localization, as demonstrated in previous studies with TMEM170A .

  • Consider the position of tags (N- versus C-terminal) based on the predicted membrane topology of TMEM170A .

• RNAi Approach Optimization:

  • Design multiple siRNA sequences targeting different regions of TMEM170A mRNA to confirm specificity of phenotypes .

  • Validate knockdown efficiency at both mRNA (qRT-PCR) and protein levels (Western blot or immunofluorescence) .

  • Use appropriate non-targeting control siRNAs that activate similar cellular pathways without targeting TMEM170A.

• Cell Type Considerations:

  • Different cell types have varying baseline ER morphologies and may respond differently to TMEM170A manipulation .

  • HeLa K cells have been successfully used in previous studies, but results should be validated in multiple cell lines .

• Quantification Methods:

  • Develop objective, automated quantification methods for analyzing changes in ER morphology to reduce observer bias .

  • Establish clear criteria for categorizing ER structures (sheets versus tubules) and nuclear envelope alterations .

• Control for Secondary Effects:

  • Monitor cell viability and proliferation, as dramatic changes in ER structure might induce ER stress responses or affect cell health .

  • Assess if observed phenotypes are direct effects or consequences of altered interactions with other proteins like RTN4 .

Attention to these methodological considerations ensures that experiments manipulating TMEM170A expression provide reliable insights into its biological functions in ER morphogenesis.

How does TMEM170A influence nuclear pore complex formation and what techniques can be used to measure this?

TMEM170A has been shown to influence nuclear pore complex (NPC) formation, with its downregulation leading to decreased density of NPCs in the nuclear envelope . To study this relationship and measure the effects of TMEM170A on NPC formation, researchers can employ several techniques:

• Immunofluorescence Microscopy:

  • Stain for NPC components using antibodies against nucleoporins (Nups) such as mAb414 (which recognizes several FG-nucleoporins) to visualize and quantify NPC distribution .

  • Use high-resolution confocal microscopy to measure changes in NPC density following TMEM170A manipulation .

  • Analyze the pattern and intensity of nuclear rim staining to assess NPC distribution and abundance.

• Super-resolution Microscopy:

  • Employ techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED), or stochastic optical reconstruction microscopy (STORM) to resolve individual NPCs that are below the diffraction limit of conventional light microscopy.

  • Quantify NPC numbers, clustering, and spatial distribution at near-molecular resolution.

• Transmission Electron Microscopy (TEM):

  • Use TEM to directly visualize NPCs as distinct structures spanning the nuclear envelope .

  • Perform morphometric analysis to count NPCs per unit length of nuclear envelope in thin sections.

• Biochemical Fractionation and Analysis:

  • Isolate nuclear envelopes and analyze the levels of nucleoporins by Western blotting .

  • Compare nucleoporin levels in whole cell lysates versus nuclear envelope fractions to assess correct targeting and incorporation into NPCs.

• Functional Assays for Nuclear Transport:

  • Measure nuclear transport efficiency using reporter constructs (NLS-GFP) to determine if changes in NPC density due to TMEM170A manipulation affect nucleocytoplasmic transport.

  • Perform selective permeabilization assays to assess nuclear envelope integrity and NPC functionality.

By combining these approaches, researchers can comprehensively characterize how TMEM170A influences NPC formation, potentially through its effects on nuclear envelope structure or through interactions with NPC assembly factors.

What is the relationship between TMEM170A and other TMEM family proteins, such as TMEM170B?

Understanding the relationship between TMEM170A and other family members, particularly TMEM170B, provides valuable insights into their functional significance:

• Expression Patterns and Disease Associations:

  • While TMEM170A has been characterized in terms of its role in ER morphology and nuclear envelope formation , TMEM170B has been studied primarily in the context of pancreatic cancer .

  • TMEM170B shows reduced expression in pancreatic adenocarcinoma (PAAD) compared to non-tumorous tissues, and this lower expression is associated with poor differentiation and worse survival outcomes .

  • A transcriptome-wide association study has linked TMEM170A expression with pancreatic cancer risk , suggesting both family members may have roles in cancer biology.

• Experimental Techniques for TMEM170B:

  • RT-PCR with specific primers has been used to measure TMEM170B expression levels in tissues (primers: forward 5′-TTCCTCTGGGCTCTCTTCTCT-3′, reverse 5′-CTGCTGCACTGGTAATCATCG-3′) .

  • Immunohistochemistry with anti-TMEM170B antibody (1:100 dilution, PA5-63072, Thermo Fisher Scientific) has been employed to assess protein expression in tissue samples .

  • Correlation and gene enrichment analysis using TCGA data has identified genes associated with TMEM170B expression .

• Research Gaps and Future Directions:

  • A comprehensive comparison of TMEM170A and TMEM170B in terms of:

    • Evolutionary conservation

    • Tissue-specific expression patterns

    • Protein-protein interactions

    • Effects on cellular structures and functions

    • Roles in disease processes

  • Would provide valuable insights into this protein family and their biological significance.

Comparative studies between TMEM170A and TMEM170B could reveal whether they have overlapping or distinct functions in cellular organization and disease processes.

What experimental procedures are required for studying recombinant TMEM170A protein production and purification?

Producing and purifying recombinant TMEM170A requires specialized techniques for membrane proteins:

• Expression System Selection:

  • For bacterial expression: Use E. coli strains optimized for membrane protein expression (e.g., C41(DE3), C43(DE3)).

  • For eukaryotic expression: Consider insect cells (Sf9, Hi5) or mammalian cells (HEK293, CHO) for proper folding and post-translational modifications.

  • Include appropriate fusion tags (His, FLAG, GST) to facilitate purification while considering TMEM170A's membrane topology .

• Construct Design:

  • Design expression constructs considering the three transmembrane domains of TMEM170A .

  • Include appropriate signal sequences for membrane targeting.

  • Consider truncated constructs containing single domains for structural studies.

  • Optimize codon usage for the chosen expression system.

• Extraction and Solubilization:

  • Extract TMEM170A from membranes using detergents suitable for multi-pass membrane proteins.

  • Screen detergents (DDM, LMNG, GDN) for optimal solubilization while maintaining protein structure and function.

  • Use mild solubilization conditions to preserve protein-protein interactions if studying complexes with partners like RTN4 .

• Purification Strategy:

  • Implement multi-step purification:

    • Affinity chromatography using tag-specific resins

    • Size exclusion chromatography to separate monomers from aggregates

    • Ion exchange chromatography for final polishing

  • Monitor protein purity by SDS-PAGE and Western blotting.

  • Verify proper folding through circular dichroism or limited proteolysis.

• Quality Control:

  • Assess protein homogeneity by analytical size exclusion chromatography.

  • Verify membrane insertion and topology using protease protection assays.

  • Confirm functionality through binding assays with known interaction partners like RTN4 .

These procedures provide a foundation for producing recombinant TMEM170A for structural and functional studies, though optimization will be required based on specific experimental needs.

How might TMEM170A function be studied in disease models relevant to ER dysfunction?

TMEM170A's critical role in ER morphology suggests potential implications for diseases involving ER dysfunction:

• Cancer Models:

  • Examine TMEM170A expression in cancer cell lines and patient samples, particularly pancreatic cancer where a transcriptome-wide association study has already linked TMEM170A expression with cancer risk .

  • Compare with TMEM170B, which shows reduced expression in pancreatic adenocarcinoma associated with poor prognosis .

  • Manipulate TMEM170A levels in cancer cell lines to determine effects on proliferation, migration, and response to ER stress-inducing chemotherapeutics.

• Neurodegenerative Disease Models:

  • Investigate TMEM170A expression and function in cellular models of neurodegenerative diseases characterized by ER stress, such as Alzheimer's or Parkinson's disease.

  • Determine if TMEM170A-mediated changes in ER structure affect the processing of disease-associated proteins.

  • Use primary neurons or iPSC-derived neurons from patients to study disease-specific alterations in TMEM170A function.

• Experimental Approaches:

  • Gene editing: Use CRISPR/Cas9 to modify TMEM170A in disease model systems.

  • High-content imaging: Develop automated analysis pipelines to quantify ER morphology changes in large sample sets.

  • Transcriptomics and proteomics: Identify global changes associated with TMEM170A dysfunction in disease contexts.

  • Functional assays: Measure ER stress responses, protein secretion efficiency, and calcium signaling in relation to TMEM170A levels.

• Therapeutic Implications:

  • Screen for small molecules that modulate TMEM170A function or its interaction with RTN4 .

  • Investigate whether altering the TMEM170A/RTN4 balance can rescue disease phenotypes in cellular or animal models.

  • Explore gene therapy approaches to normalize TMEM170A expression in conditions where it is dysregulated.

Studying TMEM170A in disease models may reveal new pathogenic mechanisms and therapeutic targets for conditions involving ER dysfunction.

What technical challenges exist in studying the molecular mechanisms of TMEM170A's effect on ER sheet formation?

Investigating the molecular mechanisms behind TMEM170A's role in ER sheet formation presents several technical challenges:

• Membrane Protein Biochemistry:

  • Extracting and maintaining TMEM170A in its native conformation during purification is difficult due to its three transmembrane domains .

  • Reconstituting purified TMEM170A into artificial membrane systems that recapitulate ER membrane properties.

  • Maintaining protein stability while removing detergents for structural studies.

• Visualization Challenges:

  • Capturing dynamic ER morphology changes in living cells requires specialized high-resolution imaging.

  • Distinguishing direct effects of TMEM170A from secondary consequences on ER structure.

  • Quantitatively measuring subtle changes in ER sheet/tubule ratios requires sophisticated image analysis algorithms .

• Interaction Studies:

  • Identifying the complete interactome of TMEM170A beyond the known interaction with RTN4 .

  • Determining if TMEM170A forms higher-order complexes with other ER-shaping proteins.

  • Distinguishing direct binding partners from proteins that simply co-localize with TMEM170A.

• Functional Analysis:

  • Isolating TMEM170A's direct effects on membrane curvature from its influence on protein localization.

  • Determining if TMEM170A has enzymatic activity or primarily acts through protein-protein interactions.

  • Establishing in vitro systems that recapitulate TMEM170A's ER-shaping activity observed in cells .

• Technical Solutions:

  • Employ cryo-electron tomography to visualize TMEM170A in its native membrane environment.

  • Develop in vitro membrane remodeling assays using giant unilamellar vesicles.

  • Use protein engineering to create TMEM170A variants with altered function for structure-function analysis.

  • Apply computational modeling to predict how TMEM170A might influence membrane curvature and organization.

Addressing these challenges will require interdisciplinary approaches combining advanced imaging, biochemistry, and biophysical techniques.