Lipocortin I has been presumed to be synthesized and secreted in response to glucocorticoids yet the amino acid sequence of lipocortin I reveals no signal sequence typically necessary for proteins to enter the secretory pathway. The translocation of lipocortin I across membranes was analyzed in a cell-free system and in Xenopus oocytes. Based on the published sequence, the cDNA of human lipocortin I was cloned and expressed in Escherichia coli. Lipocortin I was purified and used to raise monoclonal antibodies. To test whether lipocortin I is secreted in vitro, transcribed lipocortin mRNA was translated in a wheat germ cell-free system in the absence and presence of microsomal membranes. Prolactin mRNA was used as a control for translocation of newly synthesized protein into membrane vesicles. Prolactin, but not lipocortin I, was translocated into the membranes. To test for secretion of lipocortin I in vivo, Xenopus oocytes were co-injected with transcripts encoding lipocortin I and prolactin, with and without the signal sequence. Prolactin with the signal sequence was released into the medium. However, neither prolactin without a signal sequence nor lipocortin I was released. Carbonate extraction, using an integral transmembrane protein as control, revealed no evidence for membrane integration of lipocortin I. Thus lipocortin I is not a secreted protein.
