Thylakoids are membrane-bound sac-like structures within the chloroplast that serve as sites for photosynthesis. Thylakoid lumen contains many electron transport proteins and is enclosed by a thylakoid membrane rich in the light-harvesting complex. Proteins targeted to the thylakoids are transported as precursors and are sorted by the general TOC/TIC import pathway. Once the precursor reaches the stroma, stromal processing peptidases remove their transit signal and expose thylakoid signal sequences. The processed precursors use distinct mechanisms to reach the thylakoid lumen or membrane.

The first mechanism is the chloroplast signal recognition particle or cpSRP-dependent pathway that transports chlorophyll-binding proteins to the thylakoid membrane using energy from GTP hydrolysis. cpSRP proteins and FtsY receptors bind the processed precursor and stimulate GTP hydrolysis. The energy derived facilitates precursor entry through the Alb3 translocase and their subsequent insertion into the thylakoid membranes. In a second mechanism, small thylakoid membrane proteins form soluble stromal intermediates with short hydrophobic signal-like peptides that allow these proteins to fold and insert into the membrane. Once inserted, the signal-like peptides are cleaved by thylakoid processing peptidases to form the mature transmembrane protein. A third pathway employs Oxa-like integrases for inserting thylakoid membrane protein spontaneously without any energy input or targeting factors.

Some proteins targeted to the lumen employ the chloroplast secretory or cpSec pathway, which transports unfolded proteins across the thylakoid membrane. Processed precursors carrying a twin-arginine or RR motif in the N terminal domain often get folded by the stromal chaperonin Cpn60. Folded peptides are then translocated to the thylakoid lumen by the chloroplast twin-arginine or cpTat pathway. cpTat peptidases possess a basic amino acid in their C-terminal domain that helps distinguish and avoid cpSec precursors and cleaves cpTat precursors only.