Functional characterization of bacterial signal peptide OmpA in a baculovirus-mediated expression system.
Signal sequences are evolutionarily conserved and are often functionally interchangeable between prokaryotes and eukaryotes. However, we have found that the bacterial signal peptide, OmpA, functions incompletely in insect cells. Upon baculovirus-mediated expression of chloramphenicol acetyltransferase (CAT) in insect cells, OmpA signal peptide led to the cytosolic accumulation of the CAT molecules in an aglycosylated, signal-peptide cleaved form, in addition to the secretion of the glycosylated CAT. When green fluorescent protein (GFP) was used as another reporter, the GFP molecules expressed from the OmpA-GFP construct was distributed primarily in the cytosol as aggresome-like structures. These results together suggest that, subsequent to the cleavage of OmpA signal peptide in the ER, some of the processed proteins are returned to the cytoplasm. Since the prototypical insect signal peptide, melittin, did not result in this ER-to-cytosol dislocation of the reporter proteins, we proposed a model explaining the dislocation process in insect cells, apparently selective to the OmpA-directed secretory pathway bypassing the co-translational transport.
Nagamine
Toshihiro
T
RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan. tnaga@postman.riken.go.jp.
Kawasaki
Yu
Y
Iizuka
Tetsutaro
T
Okano
Keiju
K
Matsumoto
Shogo
S
Choudary
Prabhakara V
PV
D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D007313:Insecta; D008164:Luminescent Proteins; D008555:Melitten / Q000378:metabolism / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C078167:OMPA outer membrane proteins; D008555:Melitten; D015500:Chloramphenicol O-Acetyltransferase