Deletion Mutants ofChlorophyll a/bBinding Proteins Are Efficiently Imported intoChloroplasts butDoNotIntegrate into Thylakoid Membranes'

Chlorophyll a/bbinding polypeptides (CABp) areintegral thylakoid membraneproteins containing three membrane-spanning helices. We havecreated aseries ofmutations intomato CABp totestwhether individual membranehelices withhydrophilic flanking sequences, whenfusedtoa transit peptide, canbe imported intochloroplasts andcorrectly targeted tothylakoid membranes. Allofthemutated precursors, including thosewith large C-terminal andinternal deletions, wereimported successfully, showing thattheseregions ofthematureCABparenot required forimport into chloroplasts. Allmutants tested, containingeither oneortwomembranehelices, werefoundprimarily in thestroma andnotinthethylakoids. Thesmall amountofprotein foundassociated withthethylakoids waslargely resistant to alkali extraction butwassensitive toprotease, unlike wild-type protein, whichisresistant tobothtreatments. Whenincubated withthylakoids intheabsence ofstromaand/or ATP,asignificant amountofwild-type protein assumesaformthatisresistant to alkali extraction butisprotease sensitive, like theimported deletion proteins. Thisformofthewild-type protein isnotchased into aprotease-resistant formbyadding stromaand/or ATP.These results suggest thatCABpcanspontaneously associate with membranes asanaberrant species that isnotanintermediate in theprocess ofintegration. Theinability ofthedeletion formsof CABptoassumeaprotease-resistant conformation suggests that correct integration isafforded byelements within theentire protein thatcollectively contribute totheproper conformation ofthe protein. Theability ofdeletion mutants toassociate withthylakoids inanonphysiological waysuggests thatthestudy ofsuch mutantsmay notbeuseful inelucidating thylakoid-targeting signals.