MITOCHONDRIAL PROTEIN IMPORT DURING COMPROMISED OXIDATIVE PHOSPHORYLATION AND REDUCED EXTERNAL ATP 587

Abstract

Mitochondrial biogenesis requires the import of nuclear-encoded proteins. We have previously shown that skeletal muscle intermyofibrillar (IMF) mitochondria are capable of importing malate dehydrogenase (MDH) at a rate which is 3-4 fold faster than the sub-sarcolemmal (SS) mitochondria. We wished to determine if this was due to differences in mitochondrial respiration and ATP production. IMF mitochondria demonstrated a 3.8 ± 0.7-fold (n=6) higher state 3 respiratory rate, and a 2.2-fold higher affinity for ADP than the SS subfraction. These results may be linked to a greater amount of adenine nucleotide translocase (ANT) in the IMF subfraction. By establishing the dose-dependent inhibition of respiration with atractyloside (ATR), a competitive inhibitor of the ANT, we were able to closely match state 3 rates in IMF and SS mitochondria. Similar rates of state 3 respiration led to equalization of MDH import in IMF and SS mitochondria. Pre-treatment of both IMF and SS mitochondria with a high concentration (100 μM) of ATR inhibited import to 30 ± 4% (n=3) of untreated values in SS, but was less effective (58 ± 8%; p<0.05) in IMF mitochondria. Thus, MDH import into the SS mitochondria was more sensitive than the IMF subfraction to altered state 3 respiratory rates. To investigate the role of ATP outside the mitochondria in the import process, apyrase (3U) treatment was used to deplete external ATP levels. This reduced MDH import to 32 ± 4% (n=3) in SS, but to a much larger extent (9 ± 2%; p<0.05) in the IMF subfraction. These data demonstrate a greater sensitivity of the IMF subfraction to reduced external ATP concentrations. Combinations of apyrase and ATR treatment reduced MDH import dramatically, to only 6-11% of untreated SS and IMF mitochondria. These data provide evidence linking respiration and ATP production with the ability to import precursor proteins. This may be a key factor in the regulation of protein import, and may play an important role in mitochondrial biogenesis