TRIT1 Antibody

What is TRIT1 and what functional significance does it have in cellular biology?

TRIT1 belongs to the family of tRNA isopentenyltransferases (IPTases) that modify both cytoplasmic and mitochondrial tRNAs in eukaryotic cells. It catalyzes the addition of an isopentenyl group to N6 of adenosine at position 37 (i6A37) in the anticodon loop of specific tRNAs. This modification is crucial for proper codon recognition and translational efficiency and fidelity .

The biological importance of TRIT1 is underscored by the fact that pathogenic mutations in this gene cause mitochondrial insufficiency and neurodevelopmental disease, primarily due to decreased modification of the mitochondrial tRNA substrates . The proper functioning of this enzyme is particularly critical for mitochondrial protein synthesis and consequently mitochondrial function.

What is the molecular structure and characteristics of TRIT1 protein?

TRIT1 is a 467 amino acid protein with a calculated molecular weight of 53 kDa, although it is typically observed at 53-60 kDa in experimental settings . The protein contains an amino-terminal mitochondrial targeting sequence (MTS) that directs its import into mitochondria for modification of mitochondrial tRNAs .

Studies using structural modeling based on homologous yeast isopentenyltransferase have revealed that TRIT1 contains a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem . The p.Arg323 residue, which is a site of pathogenic mutation (p.Arg323Gln), is somewhat removed from the catalytic center but plays an important role in tRNA binding .

What are the recommended applications and protocols for TRIT1 antibody?

The TRIT1 antibody (e.g., 17141-1-AP) is primarily used in Western Blot (WB) and ELISA applications. For Western Blot applications, the recommended dilution is 1:500-1:1000 . It shows reactivity with human and mouse samples and has been validated in mouse placenta tissue .

Western Blot Protocol Recommendations:

• Prepare protein samples and perform SDS-PAGE separation

• Transfer proteins to a membrane

• Block the membrane with appropriate blocking buffer

• Incubate with TRIT1 antibody at 1:500-1:1000 dilution

• Wash and incubate with secondary antibody

• Develop using preferred detection method

Note that it is recommended to titrate the antibody in each testing system to obtain optimal results, as the optimal dilution may be sample-dependent .

How should TRIT1 antibody be stored and handled for maximum stability?

For optimal stability, TRIT1 antibody should be stored at -20°C, where it remains stable for one year after shipment . The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is unnecessary for -20°C storage, simplifying handling procedures . Some preparations (20μl sizes) contain 0.1% BSA to enhance stability .

When working with the antibody, avoid repeated freeze-thaw cycles and maintain cold chain management during experimental procedures to preserve antibody activity and specificity.

How does TRIT1 mitochondrial targeting mechanism function, and how can it be experimentally verified?

TRIT1 employs an N-terminal mitochondrial targeting sequence (MTS) to direct its import into mitochondria. Unlike Saccharomyces cerevisiae and Caenorhabditis elegans, which use alternative translation initiation for mitochondrial targeting of their IPTases, TRIT1 appears to utilize a different mechanism .

This mitochondrial targeting can be experimentally verified through several approaches:

• GFP fusion studies: Research has demonstrated that the first 51 amino acids of TRIT1 fused to GFP (TRIT1(1-51)-GFP) show punctate localization that colocalizes with MitoTracker, confirming mitochondrial targeting . Full-length TRIT1-GFP shows distribution in nuclei, cytosol, and some mitochondrial localization .

• Point mutation analysis: The MTS contains an amphipathic helix with critical basic residues. Point mutations R17E and R21E in this region (TRIT1(1-51:R17E/R21E)-GFP) impair mitochondrial targeting, providing evidence for the importance of these residues in mitochondrial localization .

• Subcellular fractionation: Researchers can isolate mitochondrial, cytosolic, and nuclear fractions and perform Western blotting to detect the presence of TRIT1 in different cellular compartments.

These experimental approaches provide complementary evidence for the mitochondrial targeting mechanism of TRIT1 and can be adapted to study the impact of mutations or other factors on this process.

What are the known pathogenic mutations in TRIT1 and their functional consequences?

Several pathogenic mutations in TRIT1 have been identified, with the p.Arg323Gln mutation being particularly well-characterized. The functional consequences include:

• p.Arg323Gln mutation:

  • Severe deficiency in i6A37 modification in both cytosolic and mitochondrial tRNAs

  • Impaired mitochondrial protein synthesis

  • Clinical presentation of mitochondrial insufficiency and neurodevelopmental disease

  • Located in a region that interacts with the tRNA backbone of the anticodon stem

Molecular modeling based on the co-crystal structure of homologous yeast isopentenyltransferase bound to substrate tRNA indicates that Arg323 is part of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center .

Complete complementation of the i6A37 deficiency in both cytosolic and mitochondrial tRNAs can be achieved by transduction of patient fibroblasts with wild-type TRIT1, confirming the causal role of this mutation .

How does TRIT1 exhibit substrate specificity for different tRNAs?

TRIT1 demonstrates complex substrate specificity for different tRNAs, which is governed by several determinants:

• A36-A37-A38 sequence recognition: TRIT1 primarily recognizes tRNAs with the A36-A37-A38 sequence in the anticodon loop .

• Anticodon-specific restrictions: TRIT1 exhibits clear substrate-specific restriction against certain tRNAs despite their containing the A36-A37-A38 sequence. For example, cytosolic tRNATrp with CCA anticodon is poorly modified despite having the A36-A37-A38 sequence .

• Position 32 influence: Evidence suggests that position 32 of tRNATrp CCA might serve as a conditional determinant for substrate-specific i6A37 modification .

The substrate specificity of TRIT1 for different tRNAs across species is summarized in the following table:

This table demonstrates the variable substrate specificities of IPTases across different species .

What methodologies can be employed to assess i6A37 tRNA modification status in experimental systems?

Researchers can employ several complementary approaches to assess i6A37 tRNA modification:

• i6A37-sensitive Northern blotting: This technique allows for the detection of i6A37 modification in specific tRNAs. The presence of i6A37 affects the migration pattern or hybridization properties of the tRNA, which can be detected by Northern blotting .

• tRNA-mediated suppression (TMS) assays: This functional approach tests the ability of i6A37-modified tRNAs to suppress specific mutations through enhanced codon recognition. This method has been used extensively to study tRNA biogenesis and modification, including i6A37 modification by TRIT1 .

• Mass spectrometry: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be used to directly quantify i6A37 levels in purified tRNA samples, providing a precise measurement of modification status.

• Complementation studies: Introducing wild-type TRIT1 into cells with TRIT1 mutations can restore i6A37 modification, which can be assessed by the above methods. This approach was used to demonstrate complete complementation of i6A37 deficiency in patient fibroblasts with the p.Arg323Gln mutation .

• Model organism systems: Both S. pombe and S. cerevisiae lacking endogenous IPTases can be used as experimental systems to study TRIT1 activity on various tRNA substrates .

How does TRIT1 deficiency impact mitochondrial function and protein synthesis?

TRIT1 deficiency significantly impacts mitochondrial function through several mechanisms:

• Decreased i6A37 modification in mitochondrial tRNAs: Patient cells with p.Arg323Gln TRIT1 mutation show severe deficiency in i6A37 in mitochondrial tRNAs .

• Impaired mitochondrial protein synthesis: The lack of proper tRNA modification reduces translational efficiency and fidelity in a codon-specific manner .

• Mitochondrial respiratory chain defects: Patients with TRIT1 mutations exhibit severe combined mitochondrial respiratory chain defects, indicating compromised electron transport chain function .

• Clinical manifestations: These molecular defects manifest as mitochondrial insufficiency leading to neurodevelopmental disease .

The critical nature of TRIT1 function in mitochondria is emphasized by the observation that pathophysiology of TRIT1-R323Q-associated neurodevelopmental disease is attributable primarily to deficient mitochondrial tRNA modification and consequent impairment of mitochondrial mRNA translation, even though cytosolic tRNAs are also hypomodified .

What technical considerations should be addressed when using TRIT1 antibody in complex experimental designs?

When incorporating TRIT1 antibody in complex experimental designs, researchers should consider several technical aspects:

• Subcellular fractionation purity: When studying TRIT1 in different cellular compartments, ensuring pure fractions of mitochondria, cytosol, and nucleus is essential for accurate localization studies.

• Antibody specificity validation: Confirm the specificity of TRIT1 antibody using appropriate controls:

  • TRIT1 knockout/knockdown cells

  • Cells overexpressing TRIT1

  • Peptide competition assays

• Co-immunoprecipitation considerations: When using TRIT1 antibody for co-IP to identify interaction partners:

  • Optimize antibody concentration to avoid non-specific binding

  • Include appropriate controls (IgG control, lysate input)

  • Consider crosslinking approaches for transient interactions

• Immunofluorescence optimization: For cellular localization studies:

  • Test different fixation methods (paraformaldehyde vs. methanol)

  • Optimize permeabilization conditions for mitochondrial antigen access

  • Use co-staining with established mitochondrial markers

• Western blot considerations:

  • Note the observed molecular weight range (53-60 kDa) which may vary slightly from the calculated weight (53 kDa)

  • Optimize protein extraction methods, particularly for mitochondrial TRIT1

  • Consider using gradient gels for better resolution

• Species-specific considerations: While the antibody shows reactivity with human and mouse samples , validation should be performed when using other model organisms.