Efficiency and diversity of protein localization by random signal sequences

1990 ◽  
Vol 10 (6) ◽  
pp. 3163-3173
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Protein Localization ◽  
Random Signal ◽  
Perinuclear Region ◽  
Hybrid Protein ◽  
Random Sequences ◽  
Beta Galactosidase

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

scholarly journals Efficiency and diversity of protein localization by random signal sequences.

1990 ◽  
Vol 10 (6) ◽  
pp. 3163-3173 ◽  
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Protein Localization ◽  
Random Signal ◽  
Perinuclear Region ◽  
Hybrid Protein ◽  
Random Sequences ◽  
Beta Galactosidase

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

scholarly journals Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent.

1987 ◽  
Vol 105 (6) ◽  
pp. 2905-2914 ◽  
Author(s):  
P Bird ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Influenza Virus ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Signal Sequence ◽  
Carboxypeptidase Y ◽  
Influenza Virus Hemagglutinin

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosylated 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.

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

1989 ◽  
Vol 109 (6) ◽  
pp. 2665-2675 ◽  
Author(s):  
I Sadler ◽  
A Chiang ◽  
T Kurihara ◽  
J Rothblatt ◽  
J Way ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Endoplasmic Reticulum ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Nuclear Localization ◽  
Protein Localization ◽  
Hybrid Protein ◽  
Nuclear Localization Signals ◽  
Hybrid Proteins ◽  
Cytochrome C1

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.

scholarly journals C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (1) ◽  
pp. 276-282 ◽  
Author(s):  
N Green ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Fusion Proteins ◽  
Signal Sequence ◽  
Dna Encoding ◽  
Luminal Side ◽  
Internal Signal ◽  
Histidinol Dehydrogenase ◽  
Arginine Permease ◽  
Er Membrane

We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end. These fusion proteins contain at least one of the internal signal sequences of arginine permease. When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint. The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane. These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane.

C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae

1992 ◽  
Vol 12 (1) ◽  
pp. 276-282
Author(s):  
N Green ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Fusion Proteins ◽  
Signal Sequence ◽  
Dna Encoding ◽  
Luminal Side ◽  
Internal Signal ◽  
Histidinol Dehydrogenase ◽  
Arginine Permease ◽  
Er Membrane

We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end. These fusion proteins contain at least one of the internal signal sequences of arginine permease. When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint. The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane. These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane.

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells

1989 ◽  
Vol 9 (11) ◽  
pp. 4977-4985
Author(s):  
D S Allison ◽  
E T Young
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Proteolytic Processing ◽  
Yeast Cells ◽  
Signal Peptidase ◽  
Single Amino Acid ◽  
Alpha Factor

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.

Recycling of the yeast v-SNARE Sec22p involves COPI-proteins and the ER transmembrane proteins Ufe1p and Sec20p

1998 ◽  
Vol 111 (11) ◽  
pp. 1507-1520 ◽  
Author(s):  
W. Ballensiefen ◽  
D. Ossipov ◽  
H.D. Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Hybrid Protein ◽  
Wild Type ◽  
Alpha Factor ◽  
Membrane Compartments

Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an alpha-factor-tagged Sec22 protein (Sec22-alpha) was employed. Only on reaching the late Golgi, can alpha-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-alpha is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-alpha is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-alpha is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-alpha rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-alpha and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.

scholarly journals The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in Saccharomyces cerevisiae

2020 ◽  
Vol 295 (30) ◽  
pp. 10406-10419 ◽  
Author(s):  
Akira Hosomi ◽  
Kazuko Iida ◽  
Toshihiko Cho ◽  
Hidetoshi Iida ◽  
Masashi Kaneko ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Signal Peptide ◽  
Secretory Pathway ◽  
Soluble Proteins ◽  
Signal Peptides ◽  
Yeast Mutant ◽  
The Past ◽  
Reporter Proteins

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae, a few proteins devoid of a signal peptide are still translocated into the ER and then N-glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain (ste24Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N-glycosylated in the ER in the ste24Δ strain, we identified the transcription factor Rme1 as a protein that is partially N-glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process.

scholarly journals Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli

2000 ◽  
Vol 182 (14) ◽  
pp. 4068-4076 ◽  
Author(s):  
Bradley J. Feilmeier ◽  
Ginger Iseminger ◽  
Diane Schroeder ◽  
Hannali Webber ◽  
Gregory J. Phillips
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Protein Localization ◽  
Cell Fractionation ◽  
Gene Fusions ◽  
Hybrid Protein ◽  
Hybrid Proteins ◽  
Green Fluorescent

ABSTRACT The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfpoptimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by thesec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported β-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.

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