Tic22 previously was identified as a component of the general import machin
ery that functions in the import of nuclear-encoded proteins into the chlor
oplast. Tic22 is peripherally associated with the outer face of the inner c
hloroplast envelope membrane, making it the first known resident of the int
ermembrane space of the envelope. We have investigated the import of Tic22
into isolated chloroplasts to define the requirements for targeting of prot
eins to the intermembrane space, Tic22 is nuclear-endoded and synthesized a
s a preprotein with a 50-amino acid N-terminal presequence. The analysis of
deletion mutants and chimerical proteins indicates that the precursor of T
ic22 (preTic22) presequence is necessary and sufficient for targeting to th
e intermembrane space. Import of preTic22 was stimulated by ATP and require
d the presence of protease-sensitive components on the chloroplast surface.
PreTic22 import was not competed by an excess of an authentic stromal prep
rotein, indicating that targeting to the intermembrane space does not invol
ve the general import pathway utilized by stromal preproteins. On the basis
of these observations, we conclude that preTic22 is targeted to the interm
embrane space of chloroplasts by a novel import pathway that is distinct fr
om known pathways that target proteins to other chloroplast subcompartments
.