Characterization of the Intracellular Distribution of Adenine Nucleotide Translocase (ANT) in Drosophila Indirect Flight Muscles

Abstract

Background: The high power output necessary for insect flight has driven the evolution of muscles with large myofibrils (primary energy consumers) and abundant mitochondria (primary energy suppliers). The intricate functional interrelationship between these two organelles remains largely unknown despite its fundamental importance in understanding insect flight bioenergetics. Unlike vertebrate muscle that relies on a phosphagen (creatine phosphate/creatine kinase) system to regulate high energy phosphate flux, insect flight muscle has been reported to lack mitochondrial arginine kinase (analogous to creatine kinase), a key enzyme that enables intracellular energy transport. Creatine kinase is known to interact with mitochondrial adenine nucleotide translocase (ANT) in the transfer of ADP and ATP into and out of the mitochondria. Results: Here, we use quantitative immunogold transmission electron microscopy to show that in Drosophila melanogaster indirect flight muscles (IFM), ANT is present in the mitochondria as well as throughout the myofibril. To confirm this unexpected result, we created a transgenic line that expresses a chimeric GFP-ANT protein and used an anti-GFP antibody to determine the intracellular distribution of the fusion protein in the IFM. Similar to results obtained with anti-ANT, the fusion GFP-ANT protein is detected in myofibrils and mitochondria. We confirmed the absence of arginine kinase from IFM mitochondria and show that its sarcomeric (i.e., intramyofibrillar) distribution is similar to that of ANT. Conclusions: These results raise the possibility that direct channeling of nucleotides between mitochondria and myofibrils is assisted by an ANT protein thereby circumventing the need for a phosphagen shuttle in the IFM. The myofibrillar ANT may represent a unique adaptation in the muscles that require efficient exchange of nucleotides between mitochondria and myofibrils.

Drosophila melanogaster Mitochondrial Carriers: Similarities and Differences with the Human Carriers

“…Drosophila sesB encodes two transcripts differing in their 3′UTR, which could determine a different intracellular distribution of AAC. Both transcripts share a mitochondrial localization motif, but it seems to be inaccessible in one transcript, determining an extra-mitochondrial localization of AAC [ 70 ]. According to this evidence, Drosophila AAC has also been detected in myofibrils of indirect flight muscles (IFM), suggesting the existence of a direct exchange of nucleotides between myofibrils and the mitochondria around them [ 70 , 71 ].…”

“…In the latter case, AAC forms a functional unit with mitochondrial CK, participating in the cytosolic export of phosphocreatine utilized to produce energy for muscle contraction. In Drosophila IFM, the apposition of mitochondria and myofibrils and the direct involvement of AAC compensate for the lack of a cytosolic buffering phosphagen system and a mitochondrial AK [ 70 ]. SesB was identified in a stress-sensitive mutant that showed paralysis in response to a mechanical stress [ 74 ].…”