YAE1D1 Human

What is YAE1D1 and what are its basic characteristics?

YAE1D1 is a protein-coding gene that produces Yae1 domain-containing protein 1, also known as Protein YAE1 homolog. The human recombinant protein contains 249 amino acids (226 amino acids plus a 23 amino acid His-tag at the N-terminus) with a molecular mass of approximately 27.7 kDa . The protein is encoded by the YAE1D1 gene (also known as C7orf36, GK003) with UniProt ID Q9NRH1 and Entrez Gene ID 57002 . YAE1D1 is conserved from yeast to humans, demonstrating its fundamental biological importance across species .

What is the primary function of YAE1D1 in human cells?

YAE1D1 functions as part of the cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery. Specifically, it forms a complex with ORAOV1 (the human ortholog of yeast Lto1) to create a target-specific adaptor complex. This complex is essential for the maturation of specific Fe-S proteins, particularly ribosome-associated ABC proteins like Rli1 (ABCE1 in humans) . Unlike other components of the CIA machinery that serve multiple targets, the YAE1D1-ORAOV1 complex is notable for its specificity, facilitating Fe-S cluster assembly only on select proteins rather than all cytosolic and nuclear Fe-S proteins .

How does the YAE1D1-ORAOV1 complex differ from other components of the CIA machinery?

The YAE1D1-ORAOV1 complex serves as a target-specific adaptor, unlike most CIA components which function as core machinery required for the maturation of numerous Fe-S proteins. Research using yeast models has demonstrated that while depletion of core CIA components affects multiple Fe-S proteins, depletion of Yae1 or Lto1 (the yeast homologs of human YAE1D1 and ORAOV1) specifically impairs the maturation of Rli1 without affecting other tested Fe-S proteins . This specificity distinguishes the YAE1D1-ORAOV1 complex as a unique component of the Fe-S protein biogenesis machinery that functions in a target-selective manner .

What key structural domains characterize YAE1D1?

YAE1D1 contains characteristic deca-GX3 motifs that are critical for its function. These motifs facilitate complex formation with ORAOV1/Lto1 . The protein does not contain conserved cysteine residues, which rules out the possibility that it might function as a recipient Fe-S protein itself . The protein sequence is evolutionarily conserved, sharing similarity with its yeast counterpart Yae1, particularly in the central region of the protein .

How does YAE1D1 interact with its binding partners in the Fe-S assembly pathway?

YAE1D1 participates in a chain of binding events for Fe-S cluster assembly. Based on studies of the yeast homologs, the process follows this sequence: ORAOV1 (Lto1 in yeast) uses its conserved C-terminal tryptophan to bind the CIA targeting complex, while the deca-GX3 motifs in both YAE1D1 and ORAOV1 facilitate their complex formation with each other. YAE1D1 then recruits the apo-form of target proteins (such as Rli1/ABCE1) to the assembly machinery . This interaction network creates a bridge between the generic CIA machinery and specific target proteins that cannot bind directly to the CIA targeting complex .

What is the molecular mechanism of YAE1D1-mediated Fe-S protein maturation?

The YAE1D1-ORAOV1 complex operates through a unique adaptor mechanism. When cytosolic Fe-S protein maturation is impaired (such as during depletion of early-acting CIA factors), the interaction between YAE1D1-ORAOV1 and the CIA targeting complex is significantly enhanced . This suggests a regulatory role in response to cellular Fe-S protein assembly status. The complex specifically facilitates Fe-S cluster assembly on Rli1/ABCE1, which is essential for ribosome biogenesis and translation initiation, particularly under aerobic conditions .

What expression systems are effective for producing recombinant YAE1D1?

Recombinant human YAE1D1 can be successfully expressed in E. coli expression systems. The protein is typically produced as a single, non-glycosylated polypeptide chain, often with a His-tag at the N-terminus to facilitate purification . For optimal stability and functionality, the recombinant protein is typically formulated in buffers containing components such as Tris-HCl, NaCl, glycerol, and reducing agents like DTT . When studying YAE1D1's function in complex with ORAOV1, co-expression of both proteins may provide advantages for analyzing their interactions and combined activities .

What purification methods yield high-purity YAE1D1 protein for in vitro studies?

High-purity YAE1D1 (>95% purity) can be obtained using proprietary chromatographic techniques, particularly when the protein contains an affinity tag such as a His-tag . After purification, the protein can be formulated in a stabilizing buffer (e.g., 20 mM Tris-HCl pH 8.0, 0.15 M NaCl, 20% glycerol, and 1 mM DTT) . For long-term storage, it is recommended to add carrier proteins (0.1% HSA or BSA) and to avoid multiple freeze-thaw cycles . SDS-PAGE analysis is typically used to confirm the purity of the isolated protein .

What methods are most effective for studying YAE1D1 interactions with other proteins?

Based on the research approaches described in the literature, several methods have proven effective for studying YAE1D1's interactions:

• Affinity purification coupled with immunostaining: This approach has been used to analyze interactions between Yae1 and Lto1 (the yeast homologs of YAE1D1 and ORAOV1) and their associations with CIA components .

• Systematic protein interaction approaches: These have been employed to discover YAE1D1 and ORAOV1 as binding partners of the CIA targeting complex .

• Co-expression systems: Expressing tagged versions of both YAE1D1 and ORAOV1 together has helped verify their tight interaction .

• Depletion studies: Creating regulatable promoter-containing strains has allowed researchers to study the effects of YAE1D1 depletion on Fe-S protein maturation .

What is known about YAE1D1 mutations in cancer?

Research has identified significantly mutated regions (SMRs) within the YAE1D1 promoter that were altered in approximately 9.3% of whole-exome sequencing data from melanoma samples . These mutations were confirmed in whole-genome sequencing data of melanomas (in 2 of 17 samples) . The specific 4-5 base pair mutations occurred within open chromatin sites of the YAE1D1 promoter . While these findings suggest a potential role of YAE1D1 dysregulation in melanoma, the exact functional consequences of these promoter mutations and their impact on YAE1D1 expression or function remain to be fully elucidated.

How does disruption of YAE1D1 function affect cellular processes?

Based on studies of the yeast homolog, depletion of YAE1D1 specifically impairs the maturation of the Fe-S protein Rli1 (ABCE1 in humans) . Since ABCE1 is essential for ribosome biogenesis, translation initiation, and various aspects of RNA metabolism, disruption of YAE1D1 function could potentially impact these fundamental cellular processes . Given that ORAOV1 (the binding partner of YAE1D1) is described as "cancer-related," and considering the identified mutations in YAE1D1 promoters in melanoma, there may be connections between YAE1D1 dysfunction and cancer pathogenesis that warrant further investigation .

How can researchers distinguish between the direct effects of YAE1D1 depletion and secondary effects due to impaired target protein function?

This represents a significant challenge in YAE1D1 research. Since YAE1D1 is involved in the maturation of essential proteins like ABCE1/Rli1, distinguishing direct effects from secondary consequences requires sophisticated experimental designs:

• Rescue experiments: Researchers can design complementation assays where wild-type or mutant YAE1D1 is reintroduced into depleted cells to identify which functions are directly restored.

• Temporal analysis: Using rapid depletion systems to examine the earliest effects before secondary consequences manifest.

• Target protein bypass: Creating systems where target proteins (like ABCE1) can acquire their Fe-S clusters through alternative pathways to determine which phenotypes are due to target protein dysfunction rather than direct YAE1D1 loss.

• Comparative analysis: Comparing phenotypes between YAE1D1 depletion and specific inhibition of its target proteins to identify overlapping and distinct effects.

What are the methodological considerations for studying the evolutionary conservation of YAE1D1 function?

The evolutionary conservation of YAE1D1 provides valuable research opportunities but requires careful methodological approaches:

• Cross-species complementation assays: Human YAE1D1 has been shown to replace its yeast counterpart, demonstrating functional conservation . Researchers can design similar experiments with YAE1D1 from various species to map the evolution of its function.

• Domain swap experiments: Creating chimeric proteins where specific domains from YAE1D1 homologs are exchanged can help identify which regions are functionally conserved and which have evolved species-specific functions.

• Sequence analysis with functional correlation: Comparing sequences of YAE1D1 homologs across species, especially focusing on the deca-GX3 motifs and other functional domains, can provide insights into evolutionary constraints and adaptations.

• Interactome comparison: Studying whether the protein interaction networks of YAE1D1 homologs are conserved across species can reveal evolutionary conservation or divergence of its molecular function.

What are the methodological challenges in studying YAE1D1's role in iron-sulfur cluster assembly?

Studying iron-sulfur cluster assembly presents several technical challenges:

• Oxygen sensitivity: Fe-S clusters are sensitive to oxygen, requiring specialized anaerobic conditions for certain experiments.

• Direct measurement difficulties: Directly measuring Fe-S cluster transfer and assembly is technically challenging, often requiring specialized spectroscopic methods.

• Functional redundancy: Potential overlap with other Fe-S assembly factors can complicate interpretation of depletion or knockout experiments.

• In vivo vs. in vitro discrepancies: The behavior of Fe-S assembly factors in purified in vitro systems may differ from their activity in the complex cellular environment.

• 55Fe radiolabeling techniques: This specialized approach is valuable for tracking Fe-S cluster assembly but requires appropriate facilities and safety considerations for working with radioactive materials .

What are the key unresolved questions about YAE1D1 function that require further investigation?

Several important aspects of YAE1D1 function remain to be fully elucidated:

• Complete target spectrum: While YAE1D1 is known to facilitate Fe-S cluster assembly on Rli1/ABCE1, it remains unknown whether there are other specific targets beyond those tested in existing studies.

• Regulatory mechanisms: How the activity of the YAE1D1-ORAOV1 complex is regulated in response to cellular needs or stress conditions is not fully understood.

• Structural details: The three-dimensional structure of YAE1D1, both alone and in complex with ORAOV1 and target proteins, would provide crucial insights into its mechanism of action.

• Disease relevance: The functional consequences of the identified YAE1D1 promoter mutations in melanoma, and potential involvement in other diseases, require further investigation.

• Post-translational modifications: Whether YAE1D1 function is regulated by post-translational modifications remains largely unexplored.

What novel experimental approaches could advance our understanding of YAE1D1 function?

Several innovative approaches could provide new insights into YAE1D1 biology:

• Cryo-EM studies: High-resolution structural analysis of the entire CIA machinery with YAE1D1-ORAOV1 and target proteins would illuminate the molecular mechanisms of Fe-S protein maturation.

• Proximity labeling approaches: BioID or APEX2-based proximity labeling could identify the complete in vivo interactome of YAE1D1 under various conditions.

• Single-molecule tracking: Real-time visualization of YAE1D1 dynamics could reveal its subcellular localization patterns and interaction kinetics.

• CRISPR screens: Genome-wide CRISPR screens in the context of YAE1D1 depletion could identify genetic interactions and potential redundant pathways.

• Patient-derived models: Studying YAE1D1 function in cellular models derived from patients with mutations in the YAE1D1 promoter or gene could reveal disease-relevant mechanisms.