In vitro and in organello phosphorylation of envelope proteins and phosphoglucomutase in spinach chloroplasts

In this investigation, we found that the main source of ATP necessary for envelope protein phosphorylation is generated by photophosphorylation. Indeed, seven envelope polypeptides exhibiting Mr of about 60, 51, 35, 33, 26, 14 and 13 kD were found to be fully labelled in organello in the presence of [32P]Pi and light. The majority of these phosphopolypeptides were also detected when envelopes were phosphorylated in vitro in the presence of [γ-32P]ATP. This supports the idea that the chloroplast stroma significantly supplies envelope protein-kinases with ATP in the light. Preincubation of intact chloroplasts in darkness or with the phosphate translocator inhibitor 4,4′-diisothiocianato-stilbene-2,2′-disulfonate (DIDS) strongly prevented chloroplast protein phosphorylation in the presence of [32P]Pi, thereby attesting the involvement of chloroplast ATP in the envelope protein phosphorylation process. In the dark, two chloroplast soluble polypeptides of about 94 and 67 kD were also labelled, when intact chloroplasts were fed with [32P]Pi, thus indicating that these two polypeptides are able to use dark-phosphorylation pathways, independent of the photophosphorylation process, to become phosphorylated. Our results show also that the 67 kD dark-phosphorylated protein is very likely to be a phosphoglucomutase, since its labelling was markedly reduced by the addition of exogenous glucose 1- or 6-phosphate.