Molecular mechanism of signal sequence orientation in the endoplasmic reticulum

Abstract

We have analyzed in vivo how model signal sequences are inserted and oriented in the membrane during cotranslational integration into the endoplasmic reticulum. The results are incompatible with the current models of retention of positive flanking charges or loop insertion of the polypeptide into the translocon. Instead they indicate that these N-terminal signals initially insert head-on with a cytoplasmic C-terminus before they invert their orientation to translocate the C-terminus. The rate of inversion increases with more positive N-terminal charge and is reduced with increasing hydrophobicity of the signal. Inversion may proceed for up to approximately 50 s, when it is terminated by a signal-independent process. These findings provide a mechanism for the topogenic effects of flanking charges as well as of signal hydrophobicity.

Fig. 1. Potential mechanisms of signal orientation in the translocon. (A) Retention of the more positive flanking sequence of the signal by interaction with negative charges on the cytosolic side at or near the translocon. (B) Loop insertion of the polypeptide followed by inversion of reverse signal–anchors with a more negative N-terminus. (C) Head-on insertion with subsequent inversion of cleavable signals and type II signal–anchors with a more positive N-terminal flanking sequence. For simplicity, the SRP receptor has been omitted. The signal sequence is schematically drawn as a helix; yet, its conformation is not known during targeting and reorientation.

Fig. 2. A model protein to study membrane protein topogenesis. (A) The chimeric model protein H1ΔLeu22 consists of an N-terminal signal–anchor sequence with a 230-residue C-terminal domain. It inserts in both orientations as schematically depicted (B). The C-terminal domain contains two diagnostic glycosylation sites (circles) that are core-glycosylated upon translocation into the ER lumen. (C) Upon labeling of transfected COS-1 cells with [³⁵S]methionine for 40 min, followed by immunoprecipitation using an antibody against the very C-terminal sequence (open box), SDS–gel electrophoresis and autoradiography, the glycosylated and endoglycosidase H (EH) sensitive N_cyt/C_exo forms are easily distinguished from the unglycosylated N_exo/C_cyt forms. The number of attached glycans is indicated. Cyt, cytoplasm; exo, exoplasm.

Fig. 3. Signal orientation depends on the size of the protein. (A) Schematic representation of the constructs H1ΔLeu22[#] with different C-terminal domain lengths. All constructs share the signal and the following 96 amino acids of H1ΔLeu22 as well as the C-terminal epitope for immunoprecipitation (open box). (B) The constructs were expressed in COS-1 cells, labeled with [³⁵S]methionine for 40 min, immunoprecipitated and analyzed by SDS–gel electrophoresis (14 and 10% polyacrylamide on the left and the right gel, respectively) and phosphorimaging. The positions of molecular weight markers of 15, 20, 26, 37, 50 and 64 kDa (left) and 61 and 84 kDa (right) are indicated. (C) The fraction of polypeptides with a glycosylated and thus translocated C-terminus was quantified using phosphorimager analysis. The average and standard deviation of three experiments (including the one shown in B) was plotted versus the length of the C-terminal domain.

Fig. 4. Glycosylation state reflects protein topology. (A) Alkaline extraction: COS-1 cells expressing H1ΔLeu22[110], [170], [230] or the secretory protein H20– were labeled with [³⁵S]methionine, and subjected to alkaline extraction. After centrifugation, the pellet (P) and the supernatant (S), as well as an equal aliquot of the total starting material (T) were analyzed by immunoprecipitation, SDS–gel electrophoresis, and autoradiography. (B) Protease protection: COS-1 cells expressing H1ΔLeu22[110], [170], [230] or wild-type H1 were labeled with [³⁵S]methionine, broken by swelling and scraping, and incubated with or without trypsin (Tryp) and with or without Triton X-100 (TX). Trypsin was then inhibited and the proteins were analyzed by immunoprecipitation, SDS–gel electrophoresis, and autoradiography. Since the broken cells were incubated at 4°C in the absence of protease inhibitors, some unglycosylated polypeptides were lost even in the absence of added trypsin, most likely by released lysosomal proteases. (C) Protein stability: COS-1 cells expressing H1ΔLeu22[170] or [460] were pulse-labeled for 40 min with [³⁵S]methionine and chased with excess unlabeled methionine for 0–90 min. Both pulse and chase were performed in the presence (+CHX) or absence (–CHX) of 1 µg/ml cycloheximide. The proteins were analyzed by immunoprecipitation, SDS–gel electrophoresis, and autoradiography. Upon quantitation of the glycosylated and unglycosylated products, their half-lives were calculated as indicated in minutes. The half-lives were used to correct the apparent fraction of C-terminally translocated polypeptides for degradation during the labeling period. Correction changed the values for H1ΔLeu22[170] from 36 to 31% and for H1ΔLeu22[460] from 51 to 48% in the absence of cycloheximide, and from 77 to 74% and from 73 to 71%, respectively, in the presence of cycloheximide. Protein degradation thus did not significantly distort the results.

Fig. 5. Topology depends on translation time. COS-1 cells expressing H1ΔLeu22[110] (triangles), H1ΔLeu22[170] (circles) or H1ΔLeu22 [230] (rectangles) were labeled with [³⁵S]methionine in the presence of different concentrations of cycloheximide. The labeled proteins were immunoprecipitated and analyzed by gel electrophoresis and autoradiography. Protein oriention was quantified by phosphorimager analysis and plotted as a function of cycloheximide concentration. The average with standard deviation of three determinations is shown.

Fig. 6. Signal inversion stops after ∼50 s. H1ΔLeu22[#] constructs were expressed in COS-1 cells in the presence of 1 µg/ml cycloheximide and analyzed as in Figure 3. (A) Protein orientation was quantified by phosphorimager analysis and plotted with open squares either as a function of the length of the C-terminal domain (B) or as a function of translation time (C). The elongation rate in the presence of 1 µg/ml cycloheximide was reduced by a factor of 1.8, as determined from the reduction in the total signal. For comparison, protein orientation determined in the absence of cycloheximide (from Figure 3C) is shown with filled squares. The average with standard deviation of three determinations is shown.

Fig. 7. Flanking charges and hydrophobicity determine the rate of signal inversion. Starting from the construct series H1ΔLeu22[#] (panel F), further series of proteins with increasing or decreasing hydrophobicity of the signal sequence and/or with higher or lower N-terminal positive charge were analyzed. Protein orientation was plotted versus translation time as in Figure 6C. The N-terminal amino acid sequence and the length of the oligo- leucine segment are indicated. Experiments were performed in the absence (filled squares) or presence of 1 µg/ml cycloheximide (open squares). The arrows mark the time at which signal inversion stops. Each value represents the average of at least two individual determinations.