Translocation in Yeast and Mammalian Cells: Not All Signal Sequences are Functionally Equivalent

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosytated protein is transported to the vacuole. In this paper, we show that this signal peptide does not function in mammalian cells: CPY expressed in COS-1 cells is not glycosylated, does not associate with membranes, and retains its signal peptide. In a mammalian cell-free protein-synthesizing system, CPY is not translocated into microsomes. However, if the CPY signal is either mutated to increase its hydrophobicity or replaced with that of influenza virus hemagglutinin, the resulting precursors are efficiently translocated both in vivo and in vitro. The implications of these results for models of signal sequence function are discussed. T HE signal hypothesis (Blobel and Dobberstein, 1975) provides a satisfying explanation of the targeting and translocation of newly synthesized proteins across the membrane of the endoplasmic reticulum (ER) I of eukaryotic cells. Essential to the hypothesis are the interactions between a hydrophobic segment of the secretory protein (the signal or leader) and the translocation machinery of the cell. In some circumstances, these interactions begin as the hydrophobic signal of a nascent polypeptide emerges from the ribosome and binds to signal recognition particle (SRP; Walter et al., 1984). Synthesis of the remainder of the protein may then be halted or slowed until the bound SRP attaches to its receptor on the membrane of the ER, establishing a functional ribosome-membrane junction through which the nascent polypeptide is translocated (Walter and Blobel, 1981; Walter et al., 1984; Meyer, 1985). In other cases, interaction between the signal sequence and the translocation machinery may not occur until synthesis of the polypeptide is completed, or nearly so (Ainger and Meyer, 1986; Perara et al., 1986; Mueckler and Lodish, 1986). Although the factors that determine whether translocation is coor posttranslational are not well understood, it is clear that neither process occurs in the absence of a functional signal: removal or mutation of the sequence coding for the signal results in proteins that cannot be translocated (Gething and Sambrook, 1982; Carlson and Botstein, 1982; Chao et al., 1987). Conversely, addition of a signal peptide causes proteins that normally would be sequestered on the cytoplasmic side of the ER to be transDr. Phillip Bird's current address is Commonwealth Serum Laboratories, 45 Poplar Rd, Parkville 3052, Australia. L Abbreviations used in this paper: CPY, carboxypeptidase Y; ER, endoplasmic reticulum; HA, hemagglutinin; tun, tunicamycin. located both in vivo and in cell-free systems (Sharma et al., 1985; Lingappa et al., 1984). Signal peptides have been demonstrated on secretory and membrane proteins of all organisms studied to date (for review, see Rapoport, 1986). Although there is no obvious conservation of primary amino acid sequence or length, even between signals on proteins from the same organism, statistical analyses have suggested that signals from both prokaryotes and eukaryotes are organized along similar lines (von Heijne, 1981; Perlman and Halvorson, 1983). A typical signal sequence appears to consist of three regions: a positively charged amino terminal (n) region, a central hydrophobic (h) region, and a more polar carboxy terminal (c) region, which defines the cleavage site (von Heijne, 1985). Of particular interest is the h region, since a number of studies have suggested that the overall hydrophobicity of a signal sequence is important to its function (Rapoport, 1986). In most cases, a mutation which abolishes the function of a signal peptide replaces a hydrophobic residue in the h region with a charged residue (Rapoport, 1986). Because of the structural similarities between signal sequences from organisms widely separated on the evolutionary scale, it was perhaps not surprising to find that signals from one organism can function in another. For example, the bacterial 13-1actamase signal sequence is functional in vertebrate systems in vivo and in vitro (Mueller et al., 1982; Wiedmann et al., 1984); the rat preproinsulin signal works in bacteria (Talmadge et al., 1980); and the signals of human interferon (Hitzeman et al., 1983) and influenza virus hemagglutinin (Jabbar et al., 1985) function in yeast. In addition, yeast invertase is translocated in mammalian systems both in vivo (Bergh et al., 1987), and in vitro (Perlman and Halvorson, 1981), and the precursors to several other yeast proteins such as a-factor (Julius et al., 1984) and killer toxin (Bostian 9 The Rockefeller University Press, 0021-9525/87/12/2905/10 $2.00 The Journal of Cell Biology, Volume 105 (No. 6, Pt, 2), Dec, 1987 2905-2914 2905 on July 4, 2017 jcb.rress.org D ow nladed fom et al., 1983) have been shown to translocate in mammalian in vitro systems. In this paper we show that the transfer of signal function between different organisms is not universal: a signal sequence that works efficiently in yeast is incapable of directing translocation in mammalian systems. Carboxypeptidase Y (CPY) from Saccharomyces cerevisiae contains a cleavable amino terminal signal sequence that directs it into the lumen of the yeast ER (Blachly-Dyson and Stevens, 1987; Johnson et al., 1987). However, CPY cannot be translocated across mammalian membranes either in vivo or in vitro, unless its own signal sequence is replaced by a signal from a mammalian secretory protein. We also show that mutating the CPY signal to increase its hydrophobicity-by replacing either one of its two glycine residues with a leucineallows it to direct CPY into the mammalian ER. Materials and Methods