Figure 1.
α-Helical proteins are efficiently transported through the Sec61 or SecY translocon. A and B, experimental strategy to study Sec61- and SecY-mediated translocation in eukaryotic cells and E. coli. Rectangle, signal peptide; helices, α-helical structure; straight line, intrinsic disorder. A, Sec61-mediated translocation in eukaryotic cells. Left, schematic diagram to illustrate the subcellular localization and post-translational modification of the analyzed substrates (1). Proteins containing α-helical domains are translocated through the Sec61 translocon. During translocation, the signal peptide is cleaved, and N-linked glycans are added (2). Intrinsically disordered proteins are not imported into the ER and remain unmodified in the cytosol. Right, schematic diagram of a Western blot analysis. Upon Endo H or PNGase F treatment, the glycans of proteins imported into the ER (1) are cleaved off, leading to an increased electrophoretic mobility during SDS-PAGE (3). Proteins that have not been imported into the ER contain an uncleaved signal peptide, are unglycosylated (2), and migrate slightly more slowly than unglycosylated proteins after retrotranslocation from the ER. B, SecY-mediated translocation in E. coli. Left, schematic diagram to illustrate the subcellular localization of the analyzed substrates (1). Proteins dominated by α-helical domains are translocated through the SecY translocon into the periplasm. Upon translocation, the signal peptide is cleaved (2). Intrinsically disordered proteins are not imported into the periplasm and stay in the cytosol. Respective fractions were separated, and proteins were detected afterward by Western blotting (right). C and D, top, schematic presentation of the constructs analyzed. Dark rectangle, signal peptide; α-helical structure derived from PrP is indicated by helices; polygons, N-linked glycosylation acceptor site; gray rectangle, GPI anchor signal sequence. The total length of the proteins is indicated. C, α-helical proteins are efficiently translocated through the Sec61 complex in mammalian cells and the SecY complex in E. coli. Left, cell lysates of transiently transfected HeLa cells were analyzed by Western blotting. One set of cells was cultivated in the presence of the proteasomal inhibitor MG-132 (30 μm, 3 h). To analyze N-linked glycosylation, lysates were treated with PNGase F (+) before Western blotting. White arrowhead, unglycosylated protein fraction; black arrowhead, glycosylated protein fraction. Right, transformed E. coli expressing the protein either with the DsbA or PelB signal peptide were fractionated, and the cytoplasmic (C) and periplasmic (P) fractions were analyzed by Western blotting. Unprocessed full-length constructs (dots) and constructs after signal peptide cleavage (asterisks) are marked. The cytosolic chaperonin GroEL and the periplasmic MBP were analyzed in parallel. Translocation efficiency is indicated. Data represent mean ± S.E. of at least three independent experiments. D, charged residues interfere with translocation in E. coli, but not in mammalian cells. The naturally occurring polybasic motif (amino acid sequence KKRPK) of PrPC was introduced next to the signal peptide. Expression and translocation in HeLa cells (left) and E. coli (right) was analyzed as described in C.