Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.     

Context affects nuclear protein localization in Saccharomyces cerevisiae.

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.     

Mediation, by Saccharomyces cerevisiae translocation signals, of beta-lactamase transport through the Escherichia coli inner membrane and sensitive method for detection of signal sequences.

Signal sequences of Saccharomyces cerevisiae invertase and alpha-factor pheromone were tested for the ability to mediate protein transport through the inner membrane of Escherichia coli by fusion to bacterial beta-lactamase lacking the signal sequence (blaS0). Both types of transformants exhibited ampicillin resistance in accordance with the transport of the fused protein to the periplasmic compartment. This compartment contained most of the beta-lactamase activity present in the cell. Therefore, the tested yeast signal sequences, which conferred translocation of their proteins across the membrane of the endoplasmic reticulum in S. cerevisiae, can provide the same function in E. coli. The screening for ampicillin resistance among blaS0 fusions provides a convenient method for the isolation of functional yeast and possibly higher eucaryotic signal sequences.     

Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.     

Signal-sequence Trap in Mammalian and Yeast Cells: A Comparison

Membrane associated and secreted proteins are translated as precursors containing a signal peptide that allows protein-insertion into the membrane of the endoplasmic reticulum and is co-translationally removed in the lumen. The ability of the signal peptide to direct a polypeptide into the secretory pathway is exploited in methods developed to select cDNAs encoding such proteins. Different strategies are known in which cDNA libraries can be screened for signal peptides by the ability of the latter to rescue the translocation of signal sequence-less proteins. In one method, a cDNA library is tested for interleukin 2 receptor α chain translocation to the membrane in COS cells, in another one for invertase secretion from yeast. In this work, we compared the two systems by testing six mouse signal peptides in COS and yeast cells. All of them were functional in the mammalian system, whereas only three of them in yeast. Two other sequences needed the 5′ cDNA sequence flanking the ATG codon to be removed in order to enable protein translocation. Although the structure of signal sequences and the functioning of the secretory machinery are well conserved from prokaryotes to eukaryotes, it seems evident that not all signal peptides can be interchanged between different proteins and organisms. In particular, signal peptides that are functional in the mammalian system do not necessarily lead to protein translocation in yeast. Received: 9 March 2001     

Signal peptides open protein-conducting channels in E. coli.

Plasma membrane vesicles and protoplasts of Escherichia coli were fused to planar lipid bilayers and studied with electrophysiological techniques. Large transmembrane aqueous channels were opened when 0.2 nM LamB signal peptide was added to the cytoplasmic side of the membrane. These aqueous pores are similar in conductance to those previously observed in mammalian endoplasmic reticulum when puromycin is used to release and thus unplug nascent translocating chains. Signal sequences have been previously shown to be necessary and sufficient for targeting proteins to cellular membranes. These results demonstrate that signal peptides are sufficient for opening the protein-conducting channels. We suggest that they are the physiological ligands that open protein-conducting channels at the initiation of protein translocation across prokaryotic plasma membrane and mammalian endoplasmic reticulum.     

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.     

Intragenic revertants of yeast invertase variants with secretion-defective leader sequences.

Several secretion-defective variants of invertase from Saccharomyces cerevisiae were generated by replacement of the wild-type signal sequence codons with DNA fragments with random sequences. Strains encoding these proteins failed to grow on medium containing sucrose as the sole source of carbon. The invertase that was made in these strains was found to fractionate with soluble, cytoplasmic proteins, and indirect immunofluorescence confirmed that the mutant invertase was located throughout the cytoplasm. To define the defects in the secretion-defective leader sequences, we selected revertants by requiring growth on sucrose. Surprisingly, most of the reversion events consisted of point changes and duplications in the upstream noncoding portion of the gene. Each of these changes introduced several hydrophobic residues into the nonfunctional leader sequences, suggesting that the defective random leader peptides might simply lack adequate hydrophobicity to be effective signal peptides.     

Recognition of a subset of signal sequences by Ssh1p,a Sec61p-related protein in the membrane of endoplasmic reticulum of yeast Saccharomyces cerevisiae

Ssh1p of Saccharomyces cerevisiae is related in sequence to Sec61p, a general receptor for signal sequences and the major subunit of the channel that guides proteins across the membrane of the endoplasmic reticulum. The split-ubiquitin technique was used to determine whether Ssh1p serves as an additional receptor for signal sequences in vivo. We measured the interactions between the N(ub)-labeled Ssh1p and C(ub)-translocation substrates bearing four different signal sequences. The so-determined interaction profile of Ssh1p was compared with the signal sequence interaction profile of the correspondingly modified N(ub)-Sec61p. The assay reveals interactions of Ssh1p with the signal sequences of Kar2p and invertase, whereas Sec61p additionally interacts with the signal sequences of Mfalpha1 and carboxypeptidase Y. The measured physical proximity between Ssh1p and the beta-subunit of the signal sequence recognition particle receptor confirms our hypothesis that Ssh1p is directly involved in the cotranslational translocation of proteins across the membrane of the endoplasmic reticulum.     

Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial endoglucanase by mammalian cells

It is well established that hydrophobic signal sequences direct proteins into or across the endoplasmic reticulum membrane in eukaryotes and cell membranes in prokaryotes. Although it is recognized that eukaryote proteins are efficiently secreted by bacterial systems, the export of bacterial proteins by eukaryotes has received little attention. To investigate membrane translocation of bacterial proteins by mammalian cells, the secretion of a bacterial endoglucanase (endoglucanase E) from stably transfected Chinese hamster ovary cells has been examined. We report that a functional endoglucanase is secreted when fused to prokaryote or eukaryote signal peptides. Furthermore, the endoglucanase was post-translationally modified before secretion. Data presented in this paper suggest that secretion of bacterial proteins by eukaryote cells may be a general phenomenon and infer that there are no specific requirements with respect to the origin of the signal sequences.     

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.     

A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional- lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.     

PHO5-LACZ hybrid proteins block translocation of native acid phosphatase in Saccharomyces cerevisiae

A set of protein hybrids composed of variable portions of the amino-terminal residues of the yeast phosphate-repressible acid phosphatase (product of PHO5) and an active fragment of bacterial beta-galactosidase has been constructed. When these PHO5-LACZ hybrids are expressed in a yeast strain carrying an intact chromosomal PHO5 gene, they show a size-dependent interference with the secretion of native acid phosphatase. Hybrid proteins containing approximately 50 residues of acid phosphatase do not affect secretion of native acid phosphatase. Hybrids containing greater than 200 residues of acid phosphatase reduce the amount of secreted acid phosphatase more than by 50%. The interference with secretion is specific for acid phosphatase. The hybrids do not affect secretion of invertase, and do not confer a growth-deficient phenotype on yeast. Both the hybrid proteins and acid phosphatase accumulate in non-glycosylated, membrane-bound forms which are sensitive to proteolysis from the cytoplasmic side of the membrane. The hybrids and accumulated acid phosphatase co-migrate on Percoll density gradients with markers of the endoplasmic reticulum, but not with markers of the Golgi or secretory vesicles. These results suggest that PHO5-LACZ hybrid proteins specifically block secretion of native acid phosphatase by interfering with enzyme after targeting but before translocation across the endoplasmic reticulum.     

Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport.

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.     

Peptide signals encode protein localization

Many bacterial proteins are localized to precise intracellular locations, but in most cases the mechanism for encoding localization information is not known. Screening libraries of peptides fused to green fluorescent protein identified sequences that directed the protein to helical structures or to midcell. These peptides indicate that protein localization can be encoded in 20-amino-acid peptides instead of complex protein-protein interactions and raise the possibility that the location of a protein within the cell could be predicted from bioinformatic data.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule.