The post-translational transport of cytoplasmically synthesized precur
sor proteins into chloroplasts requires proteins in the envelope membr
anes. To identify some of these proteins, label transfer cross-linking
was performed using precursor to the small subunit of ribulose-1,5-bi
sphosphate carboxylase (prSSU) that was blocked at an early stage of t
he transport process. Two envelope proteins were identified: an 86-kD
protein and a 75-kD protein, both present in the outer membrane. Label
ing of both proteins required prSSU and could not be accomplished with
SSU lacking a transit peptide. Labeling of the 75-kD protein occurred
only when low levels of ATP were present, whereas labeling of the 86-
kD protein occurred in the absence of exogenous ATP. Although both lab
eled proteins were identified as proteins of the outer envelope membra
ne, the labeled form of the 75-kD protein could only be detected in fr
actions containing mixed envelope membranes. Based on these observatio
ns, we propose that prSSU first binds in an ATP-independent fashion to
the 86-kD protein. The energy-requiring step is association with the
75-kD protein and assembly of a translocation contact site between the
inner and outer membrane of the chloroplastic envelope.