Recombinant Mouse Transmembrane protein 231 (Tmem231)

What is mouse Tmem231 and what is its primary function in cellular biology?

Tmem231 is a two-pass transmembrane protein that functions as a core component of the Meckel syndrome (MKS) complex at the ciliary transition zone, which is located between the basal body and axoneme . The primary function of Tmem231 is to regulate the localization of ciliary membrane proteins, acting as a gatekeeper for protein entry into cilia . Tmem231 is evolutionarily conserved from C. elegans to mammals, suggesting its fundamental importance in ciliary biology .

Research has demonstrated that Tmem231 is critical for organizing the MKS complex and maintaining proper ciliary composition . When Tmem231 function is disrupted, the localization of key ciliary proteins including Arl13b and Inpp5e is compromised, resulting in developmental abnormalities characteristic of ciliopathies .

What are the known protein interactions of mouse Tmem231?

Mouse Tmem231 interacts with multiple components of the MKS complex. Mass spectrometric analysis following localization and affinity purification (LAP) has confirmed that Tmem231 directly interacts with B9d1 and additionally associates with multiple other components of the MKS complex, including:

• Mks1

• Tctn1

• Tctn2

• Tctn3

• Cc2d2a (Mks6)

• Tmem17

These interactions have been validated through coimmunoprecipitation experiments using epitope-tagged versions of Tmem231, B9d1, and Mks1 . The interaction between Tmem231 and B9d1 appears to be particularly important, as these proteins are interdependent for localization to the transition zone .

What expression systems are optimal for producing recombinant mouse Tmem231?

For recombinant expression of mouse Tmem231, mammalian expression systems are generally preferred over bacterial or insect cell systems due to the requirement for proper post-translational modifications and membrane insertion. Based on published research approaches:

• Mammalian cell expression: HEK293T or IMCD3 cells transfected with expression vectors containing epitope-tagged Tmem231 (such as FLAG or LAP-tagged constructs) have been successfully used .

• Expression vectors: Vectors containing CMV promoters that provide high expression levels in mammalian cells are commonly used for Tmem231 expression.

• Affinity tags: For purification and detection purposes, Tmem231 has been successfully expressed with tags such as:

  • FLAG tag

  • Localization and Affinity Purification (LAP) tag (consisting of GFP and hexahistidine tags)

When expressing recombinant Tmem231, it's important to consider its transmembrane nature, which may require detergent solubilization for extraction from membranes if purification is required.

What techniques are most effective for studying Tmem231 localization and function?

Several complementary techniques have proven effective for studying Tmem231:

• Fluorescence microscopy: Immunofluorescence using antibodies against endogenous Tmem231 or against epitope tags on recombinant Tmem231 has been used successfully to localize the protein to the transition zone .

• Biochemical approaches:

  • Coimmunoprecipitation to identify protein interactions

  • Mass spectrometry to identify interaction partners

  • Western blotting to assess protein expression levels

• Genetic approaches:

  • Knockout mice (Tmem231−/−) to study in vivo function

  • siRNA or CRISPR-Cas9 for in vitro knockdown/knockout studies

• Functional assays:

  • Assessment of ciliary protein localization (e.g., Arl13b and Inpp5e) by immunofluorescence

  • Ciliary length measurements

  • Hedgehog signaling assays (since ciliary defects affect this pathway)

How does Tmem231 contribute to transition zone architecture and function?

Tmem231 is essential for the structural organization and function of the ciliary transition zone (TZ). Research has revealed several key aspects of its contribution:

• TZ assembly: Tmem231 and B9d1 are mutually dependent for localization to the TZ. In Tmem231−/− cells, B9d1 fails to localize to the TZ, and conversely, in B9d1−/− cells, Tmem231 fails to localize to the TZ .

• MKS complex organization: Tmem231 is required for the localization of other MKS complex components, such as Mks1, to the TZ .

• Ciliary membrane composition: Tmem231 functions as part of a molecular gate that regulates protein entry into cilia. In Tmem231−/− cells, the localization of membrane-associated proteins like Arl13b and Inpp5e to cilia is disrupted .

• Evolutionary conservation: The role of Tmem231 in TZ formation and function appears to be conserved from C. elegans to mammals, underscoring its fundamental importance in ciliary biology .

These findings suggest that Tmem231 plays a critical structural role in organizing the MKS complex at the TZ, which in turn controls ciliary membrane composition and signaling.

What phenotypes are observed in Tmem231-deficient experimental models?

Tmem231 knockout mice exhibit a range of phenotypes characteristic of ciliopathies, particularly Meckel syndrome. These include:

• Embryonic lethality: On a C57BL/6 background, homozygous Tmem231 mutants die around embryonic day 15.5. On a mixed C57BL/6-CD1 background, they survive until birth .

• Developmental abnormalities:

  • Kidney cysts, predominantly at the corticomedullary border

  • Hepatic ductal plate malformations

  • Hindlimb preaxial polydactyly

  • Microphthalmia (small eyes)

• Cellular and molecular defects:

  • Disrupted localization of ciliary membrane proteins, including Arl13b and Inpp5e

  • Abrogated Hedgehog signaling

These phenotypes closely resemble those seen in human Meckel syndrome, making Tmem231 mutant mice a valuable model for studying the pathogenesis of ciliopathies.

What are the known disease-associated mutations in TMEM231 and how do they affect protein function?

Several mutations in human TMEM231 have been identified in patients with orofaciodigital syndrome type 3 (OFD3) and Meckel syndrome (MKS). Key mutations and their functional consequences include:

• p.Pro125Ala mutation:

  • Found in multiple families of northern European descent

  • Compromises Tmem231 protein stability

  • Fails to rescue B9d1 localization to the TZ in Tmem231−/− cells

• p.Asn90Ile mutation:

  • Reduces Tmem231 protein levels

  • Fails to restore B9d1 localization to the TZ

  • When expressed in wild-type cells, can mislocalize B9d1 away from the TZ to the centrosome, suggesting a potential dominant-negative effect

• Other identified mutations:

  • Various truncating and missense mutations have been identified in MKS patients

  • All tested disease-associated mutations fail to fully restore the localization of ciliary proteins like Arl13b

Interestingly, although some mutations destabilize the Tmem231 protein, all tested mutant forms retain the ability to interact with B9d1, suggesting that the pathogenic mechanism may be separate from their capacity to form protein-protein interactions .

Table 1: Selected TMEM231 mutations identified in human ciliopathy patients

What approaches can be used to analyze the functional consequences of Tmem231 mutations?

Several complementary approaches have been used to analyze how mutations affect Tmem231 function:

• Biochemical approaches:

  • Coimmunoprecipitation assays to test if mutant forms of Tmem231 can interact with known partners like B9d1

  • Western blotting to assess protein stability and expression levels

• Cellular localization studies:

  • Immunofluorescence microscopy to determine if mutant Tmem231 properly localizes to the TZ

  • Analysis of whether mutant Tmem231 can rescue the localization of other TZ proteins (like B9d1) in Tmem231−/− cells

• Functional rescue experiments:

  • Testing whether expression of mutant Tmem231 can restore the localization of ciliary membrane proteins like Arl13b in Tmem231−/− cells

  • Assessing if mutant Tmem231 can rescue ciliary signaling defects

• Structure-function analysis:

  • Targeted mutagenesis to identify critical residues/domains for Tmem231 function

  • Chimeric protein approaches to identify functional domains

These approaches collectively provide a comprehensive assessment of how disease-associated mutations compromise Tmem231 function at the molecular and cellular levels.

How can animal models of Tmem231 dysfunction contribute to understanding ciliopathy pathogenesis?

Tmem231 mutant mice serve as valuable models for understanding the pathogenesis of ciliopathies, particularly Meckel syndrome and orofaciodigital syndrome. These models can be utilized in several ways:

• Developmental studies:

  • Tracking when and how developmental abnormalities first appear during embryogenesis

  • Identifying affected cell types and tissues

  • Understanding the relationship between ciliary defects and tissue-specific phenotypes

• Therapeutic testing:

  • Testing potential therapeutic approaches that might restore proper ciliary function

  • Identifying pathways that could be targeted to ameliorate disease phenotypes

• Tissue-specific knockout studies:

  • Conditional knockout of Tmem231 in specific tissues to dissect its function in different contexts

  • Understanding the temporal requirements for Tmem231 function during development

• Genetic interaction studies:

  • Crossing Tmem231 mutants with mice carrying mutations in other ciliary genes to identify genetic modifiers

  • Understanding the relationship between different ciliopathy genes and potential redundancy in function

Such studies could provide insights not only into the basic biology of cilia but also into the pathogenic mechanisms underlying human ciliopathies.

What are the challenges in studying recombinant Tmem231 and how can they be addressed?

Studying recombinant Tmem231 presents several challenges common to transmembrane proteins:

• Expression and purification:

  • Transmembrane proteins can be difficult to express at high levels

  • Solution: Use mammalian expression systems with strong promoters; optimize codon usage; consider fusion partners that enhance expression

  • Membrane extraction requires detergents that may affect protein folding/function

  • Solution: Screen multiple detergents; consider native membrane isolation techniques; use gentle solubilization conditions

• Structural studies:

  • Transmembrane proteins are challenging for structural biology approaches

  • Solution: Consider cryo-EM; use antibody fragments to stabilize structure; attempt crystallization of soluble domains

• Functional reconstitution:

  • Challenging to reconstitute transmembrane protein function in vitro

  • Solution: Consider liposome reconstitution; cell-free expression systems; membrane mimetics like nanodiscs

• Detecting protein-protein interactions:

  • Membrane environment can affect interaction properties

  • Solution: Use proximity labeling approaches (BioID, APEX); split-reporter systems; in-cell crosslinking

• Assessing dynamics and trafficking:

  • Difficult to track real-time protein movement

  • Solution: Live-cell imaging with fluorescent tags; photo-switchable tags; pulse-chase approaches

Addressing these challenges requires a combination of techniques and often necessitates collaboration between researchers with expertise in different methodological approaches.