Protein targeting towards the thylakoid lumen of chloroplasts: Proper localization of fusion proteins is only observed in vivo

Routeing of fusion proteins to the thylakoid lumen of the chloroplast was compared in vitro and in vivo. The Escherichia coli protein β-lactamase was used as a passenger to study this intraorganellar sorting process. The first step, translocation of β-lactamase into the chloroplast stroma, occurs properly both in vitro and in vivo and is dependent on the presence of a transit peptide in the protein construct. The second step, targeting towards the thylakoid lumen, is more complicated as was also observed previously when other passenger proteins were used. In vitro, the presence of a thylakoid transfer domain is not enough for routeing and proper processing. Only when the complete thylakoid lumen precursor plastocyanin was fused to β-lactamase was the fusion protein processed adequately, but routeing was still incomplete. However, in vivo, the information present in the thylakoid transfer domain was the only requirement for proper transport towards the thylakoid lumen. These data show that in vivo, the only requirement for targeting of passenger proteins towards the thylakoid lumen is the presence of a transit peptide and a thylakoid transfer domain. Furthermore, we demonstrate that the in vitro import system does not necessarily reflect the in vivo situation with respect to intraorganellar sorting.