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MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic
E C Gillman1, L B Slusher, N C Martin
1Department of Biological Chemistry, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Abstract:
MOD5 is one of several genes that code for enzymes found in mitochondria and another cellular compartment. Like other such genes, it contains two in-frame ATGs that could be used to produce two proteins, differing from each other by an amino-terminal extension. Certain other genes produce heterogeneous mRNAs with some 5' ends falling upstream of the longest open reading frame and some 5' ends falling between the first and second ATGs. In these cases, selection of transcription start sites appears to play a significant role in translation start site selection. MOD5, in contrast, produces mRNAs with 5' ends that all fall upstream of both ATGs. To determine how MOD5 encodes isozymes that are located in different cellular compartments and to determine whether they differ in structure, we constructed MOD5 and MOD5-COXIV fusions with mutations of the first, second, or both ATGs. The effect of these alterations on protein production, tRNA modification, and cellular location was assessed. Both the first and second ATGs are used to produce MOD5 protein in vivo, but only the long form of the protein is imported into mitochondria. Thus, the first 11 amino acids present on the amino-terminal extended protein are necessary for mitochondrial import. Surprisingly, this extension does not promote complete import of the long form of the protein, but rather a functional pool of the extended protein remains in the cytoplasm. The amino-terminal extension is also unusual because it is probably not proteolytically removed upon import and therefore does not constitute part of a mitochondrial presequence.
Insights
The MOD5 gene produces two proteins via two start codons. The longer protein form, with an 11-amino acid extension, is necessary for mitochondrial import but also remains functional in the cytoplasm.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The MOD5 gene encodes enzymes functioning in both mitochondria and cytoplasm.
- Like other genes, MOD5 has two in-frame ATGs, potentially producing two protein isoforms with different amino-terminal extensions.
- Unlike some genes where transcription start site selection dictates translation, MOD5 mRNAs initiate upstream of both ATGs.
Purpose of the Study:
- To investigate how MOD5 encodes isozymes targeted to different cellular compartments.
- To determine the structural differences between MOD5 protein isoforms.
- To elucidate the role of the two ATGs in MOD5 protein production and localization.
Main Methods:
- Construction of MOD5 and MOD5-COXIV fusion proteins with mutations in the first, second, or both ATGs.
- Assessment of protein production, tRNA modification, and cellular localization.
- Analysis of mitochondrial import and cytoplasmic retention of MOD5 protein variants.
Main Results:
- Both the first and second ATGs are utilized for MOD5 protein synthesis in vivo.
- Only the longer protein isoform, containing an amino-terminal extension, is imported into mitochondria.
- The 11-amino acid amino-terminal extension is essential for mitochondrial import but also leads to a functional pool remaining in the cytoplasm.
- This extension likely functions without proteolytic removal, differing from typical mitochondrial presequences.
Conclusions:
- MOD5 utilizes dual start codons to generate distinct protein isoforms with differential subcellular localization.
- The amino-terminal extension dictates mitochondrial import but also mediates cytoplasmic function, suggesting a dual-role mechanism.
- The MOD5 protein's import mechanism is unique, as the extension is not cleaved and a portion remains cytoplasmic.