RUPTURE OF THE MITOCHONDRIAL OUTER-MEMBRANE IMPAIRS PORIN ASSEMBLY

Outer membranes isolated from yeast mitochondria were capable mediatin g the in vitro insertion of porin. As with the outer membrane of intac t mitochondria, the insertion was ATP-dependent, and the inserted pori n was resistant to trypsin treatment after detergent solubilization. H owever, the extent of porin insertion into isolated outer membranes wa s much less per mg of outer membrane protein than with intact mitochon dria. The greater efficiency of intact mitochondria was not due to con tact site-mediated translocation as isolated contact sites were less a ble to insert porin than isolated outer membranes, and blockade of the contact site channel in intact mitochondria did not affect porin inse rtion. However, mitochondria that had been subjected to osmotic shock sufficient to rupture the outer membrane and deplete the contents of t he intermembrane space (i.e. mitoplasts) lost most of their ability to insert porin. Since outer membranes are isolated from mitoplasts, the low insertion activity of mitoplasts explains the low efficiency of i nsertion into isolated outer membranes. These results also indicate th at, unlike proteins that are imported to the inner membrane and matrix of the mitochondria, porin's assembly is severely reduced by breachin g the outer membrane and depletion of the intermembrane space contents .