Abstract
The human protein MDDX28 is a putative RNA helicase and a nucleocytoplasmic shuttling protein also localized to the mitochondria. Its localization is novel among RNA helicases. We have studied its intracellular targeting signals and show that the first 20 amino acids of MDDX28 are necessary and sufficient for both mitochondrial import and nuclear export of the protein. Mutation of the five leucines in the sequence to alanines abolished the mitochondrial targeting signal as well as greatly reducing the nuclear export signal, indicating that these signal sequences are highly overlapping. Two short stretches of basic amino acids separated by 44 residues were both necessary and sufficient for full nuclear localization. However, they were not absolutely essential, because the protein was present in 7% of the nuclei when both signals were mutated. This indicates that MDDX28 contains another unidentified weak nuclear localization signal(s). Three basic domains in the N-terminal half of the protein and its RNA binding ability were essential for nucleolar localization as well as transcription-inhibition-dependent localization to nuclear subcompartments. Two of these basic domains were the same as those constituting the nuclear localization signal, suggesting that they are responsible for bringing the protein into the nucleus to the sites of RNA binding. Our results indicate that MDDX28 nucleo-cytoplasmic shuttling is dependent on the availability of nascent RNA.
Highlights
Form a large functional domain involved in hydrolyzing nucleoside triphosphate, mainly ATP, and in coupling the hydrolysis to the unwinding process
Mitochondrial Targeting Signal (MTS)—Both endogenous MDDX28 and protein fused to EGFP are localized to the mitochondria of all cells, implying that it may be constantly needed in the organelle, e.g. in connection with the continuous mitochondrial transcription [1]
We had previously predicted that a MTS was located at amino acids 3–18 of MDDX28 [1] because this sequence has the MTS characteristics consisting of 16 amino acids, potentially forming an amphiphilic ␣-helix with a net positive charge
Summary
Form a large functional domain involved in hydrolyzing nucleoside triphosphate, mainly ATP, and in coupling the hydrolysis to the unwinding process (for a review, see Ref. 5). MDDX28 was recently shown to contain an RNA-dependent ATP-hydrolyzing activity [1]. Endogenous MDDX28 is localized in the mitochondria and the nucleus, where it is found in the nucleolus. Much of the intracellular transport goes through large protein complexes in the intracellular membranes, e.g. of the nucleus and mitochondria, and is largely an active process. Research on localization signals has revealed that there is no simple consensus signal for any of the intracellular compartments, suggesting that different transporter and control mechanisms apply. Proteins that localize to mitochondria and nucleus regulate the localization through formation of different isoforms by alternative splicing or by alternative transcription or translation initiation (6 –10). The aim of the present study was to identify the targeting determinants of MDDX28 as a step toward understanding the function of the protein as well as the control mechanisms for the intracellular localization
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