Topology of IEP110, a component of the chloroplastic protein import machinery present in the inner envelope membrane

Proteins from both the inner and outer envelope membranes are engaged in the recognition and translocation of precursor proteins into chloroplasts, A 110 kDa protein of the chloroplastic inner envelope membrane was identified as a component of the protein import apparatus by two methods, First, this protein was part of a 600 kDa complex generated by crosslinking of precursors trapped in the translocation process. Second, solubilization with detergents of chloroplasts containing trapped precursors resulted in the identification of a complex containing both radiolabeled precursor and IEP110, Trypsin treatment of intact purified chloroplasts was used to study the topology of IEP110. The protease treatment left the inner membrane intact while simultaneously degrading domains of inner envelope proteins exposed to the intermembrane space, About 90 kDa of IEP110 was proteolitically removed, indicating that large portions protrude into the intermembrane space, Hydropathy analysis of the protein sequence deduced from the isolated cDNA clone in addition to Western blot analysis using an antiserum of IEP110 specific to the N-terminal 20 kDa, suggests that the N-terminus serves to anchor the protein in the membrane, We speculate that IEP110 could be involved in the formation of translocation contact sites due to its specific topology.