Expression of human recombinant lipocortin I in a wheat-germ cell-free system and Xenopus oocytes. Lipocortin is not secreted.
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.
Frey
B M
BM
Department of Physiology, University of California, San Francisco.
Frey
F J
FJ
Lingappa
V R
VR
Trachsel
H
H
D000818:Animals; D017302:Annexins; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009865:Oocytes / Q000378:metabolism*; D010920:Placenta / Q000737:chemistry; D010944:Plants / Q000378:metabolism*; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics*; D014158:Transcription, Genetic; D014908:Triticum; D014981:Xenopus
D017302:Annexins; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA