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. 2004 Mar;15(3):1470-8.
doi: 10.1091/mbc.e03-08-0599. Epub 2003 Dec 10.

Sec61p contributes to signal sequence orientation according to the positive-inside rule

Veit Goder  1 , Tina Junne, Martin Spiess
Affiliations

Sec61p contributes to signal sequence orientation according to the positive-inside rule

Veit Goder et al. Mol Biol Cell. 2004 Mar.

Abstract

Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated. Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the "positive-inside rule." We tested the role of conserved charged residues of Sec61p, the major component of the translocon in Saccharomyces cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis by using diagnostic model proteins. Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences. These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal. For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides. Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.

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Figures

Figure 1.
Figure 1.
Model substrates to detect changes in signal orientation. (A) The N-terminal sequence of [Leu16](-3)CPY with a signal-anchor of 16 consecutive leucine residues (boxed), positive N- and net negative C-terminal flanking regions is shown. Diamonds indicate the positions of mutations R4E and E30K that change the charge difference Δ(C-N) from -3 to -1. Cells with wild-type Sec61p expressing [Leu16](-3)CPY were labeled with [35S]methionine for 5 min. The protein was immunoprecipitated and analyzed by SDS-gel electrophoresis and autoradiography (lane 1). Three products with zero, two, or three glycans (arrows) were produced, as confirmed by endoglycosidase H (EndoH) sensitivity (lane 2). In a protease protection experiment (lanes 3-5), labeled cells were homogenized and incubated without protease (-), with trypsin (T), or with trypsin in the presence of detergent (TD), before immunoprecipitation and analysis. Protected glycosylated products and trypsin-sensitive unglycosylated products correspond to Ncyt/Cexo and Nexo/Ccyt orientations, respectively, as illustrated schematically on the right. The C-terminal box indicates the HA-tag. Reduced charge difference by mutations R4E (lane 6) or E30K (lane 7) resulted in an increased unglycosylated population with Nexo/Ccyt orientation. (B) The internal signal-anchor sequence of 40[Leu16](+5)CPY (residues 32-74) are shown. Diamonds indicate the positions of mutations D34R and K65E that reduce the charge difference Δ(C-N) from +5 to +3. 40[Leu16](+5)CPY was expressed in cells with wild-type Sec61p and subjected to endoglycosidase H sensitivity and protease protection assays as in A. Reduced charge difference by mutations D34R (lane 6) or K65E (lane 7) resulted in an increased glycosylated population with Ncyt/Cexo orientation. The positions of molecular weight markers are indicated to the right of the autoradiographs. Cyt, cytoplasm; exo, exoplasm.
Figure 3.
Figure 3.
Specific charge mutations in Sec61p affect the orientation patterns of [Leu16](-3)CPY. (A) [Leu16](-3)CPY was expressed in Sec61p wild-type and mutant strains, pulse-labeled with [35S]methionine for 5 min, immunoprecipitated and analyzed by gel electrophoresis and autoradiography. The mutant R225E/K226E/K228E/K229E/R230E is abbreviated as R225E/... /R230E. (B) The fractions of glycosylated products (Ncyt/Cexo orientation) were quantified by PhosphorImager analysis. The average and SD of at least three individual determinations were plotted. The white bars show the orientation patterns of the mutated model proteins with wild-type Sec61p (see Figure 1A, lanes 6 and 7). Asterisks indicate a significant difference to values obtained with wild-type cells according to Student's t test (p ≤ 0.05). (C) Membrane integration of [Leu16](-3)CPY in cells expressing wild-type Sec61p and selected mutants was tested by alkaline extraction (lanes 1-15). Supernatant (S) and membrane pellet (P), as well as starting material (T, total) were analyzed by immunoblotting using anti-HA antibody. As a soluble control protein, CPY was analyzed in parallel (lanes 16-18). The positions of molecular weight markers (in kilodaltons) are indicated.
Figure 5.
Figure 5.
Product stability and glycosylation efficiency in Sec61p mutant strains. (A) To test glycosylation efficiency, the model protein 60[H1](+1) with an N-terminal portion of yeast Ste2p that contains a glycosylation site was expressed in strains with selected Sec61p mutants, labeled for 5 min with [35S]methionine and immunoprecipitated (lanes 1-9). The number of attached glycans is indicated. In addition, the glycosylation efficiency for endogenous Gas1p was analyzed in cells with different Sec61p mutants by labeling for 3 min with [35S]methionine and immunoprecipitation (lanes 10-15). An aliquot from wild-type cells was subjected to deglycosylation by endoglycosidase H (endoH) before analysis (lane 10). (B) [Leu16](-3)CPY was expressed in Sec61p wild-type and mutant strains, pulse-labeled for 5 min with [35S]methionine and chased in the presence of excess unlabeled methionine for 0-40 min, before immunoprecipitation and analysis. The apparent half-lives are >90 min for the combined glycosylated forms and 30-40 min for the unglycosylated form in all strains. Note the gradual shift of two- to threefold glycosylated form during the chase period.
Figure 2.
Figure 2.
Conserved residues of Sec61p potentially involved in decoding the flanking charges of signal sequences. The amino acid sequence and membrane topology of S. cerevisiae Sec61p is shown. Charged residues opposed to the positive-inside rule and well conserved among Sec61p orthologues are represented by filled circles.
Figure 4.
Figure 4.
The Sec61p mutants R67E, R74E, and E382R inversely affect orientation of 40[Leu16](+5)CPY, a substrate with inverted flanking charges. (A and B) Insertion patterns of 40[Leu16](+5)CPY in Sec61p wild-type and mutant strains were determined and quantified as for [Leu16](-3)CPY in Figure 3, A and B. The positions of molecular weight markers (in kilodaltons) are indicated. (C and D) Additional Sec61p mutants, D168R/E169R, E266R, and E382R, were analyzed for orientation effects for the substrates [Leu16](-3)CPY and 40[Leu16](+5)CPY.
Figure 6.
Figure 6.
Combining charge mutations R67E, R74E, and E382R. (A and B) Insertion patterns of [Leu16](-3)CPY or 40[Leu16](+5)CPY in strains with combined Sec61p mutations were determined and quantified as in Figure 3, A and B. For comparison, the values for protein orientation in single mutants have been included.
Figure 7.
Figure 7.
Sec61p mutations affect signal orientation both by charge effects and by structural effects. Insertion patterns of 40[Leu16](+7)CPY (A and B) and of 40[Leu16](+3)CPY (C and D) in Sec61p wild-type and mutant strains were determined and quantified as in Figure 3 (A and B). 40[Leu16](+7)CPY corresponds to 40[Leu16](+5)CPY in which the charge difference was increased by mutation E72K. In 40[Leu16](+3)CPY, the charge difference was reduced by mutation K65E.
Figure 8.
Figure 8.
Effects of mutations R67E, R74E, and E382R in Sec61p are independent of Ssh1p. Insertion patterns of 40[Leu16](+5)CPY and 40[Leu16](+7)CPY in strains lacking Ssh1p were determined and quantified as in Figure 3, A and B. The average of two independent experiments is plotted. For comparison, the values determined in the presence of Ssh1p in previous figures are indicated by light gray bars.
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