The N-terminal cleavable extension of plant carrier proteins is responsible for efficient insertion into the inner mitochondrial membrane

A subset of mitochondrial carrier proteins from,plants contain a cleavable N-terminal extension. We have used a reconstituted,protein import assay system into intermembrane space-depleted mitochondria to study the role of the cleavable extension in the carrier import pathway. Insertion of carrier proteins into the inner membrane can be stimulated.:by the addition of a soluble intermembrane space fraction isolated, from plant mitochondria., Greater stimulation of import of. the adenine nucleotide carrier (ANT) and phosphate carrier (Pic), which contain N-terminal cleavable extensions, was observed compared to the import of the oxglutarate malate carrier. (OMT), which does not contain a cleavable extension: Removal of the N-terminal cleavable extension from ANT and Pic: resulted in loss of stimulation of insertion into the inner membrane. Conversely, addition of the N-terminal extension from ANT or Pic to OMT resulted in significantly enhanced insertion into the inner membrane. The polytopic inner membrane proteins TIM17 and TIM23 that are imported via the carrier import pathway contain no cleavable extension, displayed high-level stimulation of insertion into the inner membrane by addition of the intermembrane space fraction. Addition of the N-terminal cleavable extension from carrier proteins to TIM23 enhanced insertion of TIM23 into the inner membrane even in the absence of the soluble intermembrane space fraction. Together, these results demonstrate that the cleavable N-terminal extensions present on carrier proteins from plants are required for efficient insertion into the inner mitochondrial membrane, and that they can stimulate insertion of any carrier-like protein into the inner membrane. (c) 2005 Elsevier Ltd. All rights reserved.