N-glycosylation of mouse Syt II. (A) Domain structure of mouse Syt II and amino acid sequences around the potential glycosylation site within the N-domain. The closed box indicates the H-region. The numbers indicate those of the amino acid residues. The glycosylation site (underlined) was disrupted by a single mutation of T34A (double underlined). (B) The wild-type and mutated molecules were expressed in the reticulocyte lysate cell-free system in the absence (−) or presence (+) of RM. After the translation reaction, an aliquot was treated with EndoH under the denaturing condition (+ lane). (C) Both molecules were expressed in COS7 cells. The cells were pulse-labeled and then the Syt II molecules were immunoprecipitated with rabbit antibody against the C-domain of Syt II. Aliquots of the immunoprecipitants were treated with EndoH (+ lanes).

Positive charges following the H-region are essential for SA-I topogenesis. (A) Construct in which eight lysine residues were mutated to neutral ones. Potential glycosylation sites are indicated; one within the N-domain and three within the cytoplasmic domain. (B) The mutant was expressed in vitro. After the translation reaction, aliquots were treated with EndoH, and with ProK in the presence (+) or absence (−) of detergent (Triton X-100). One, two, and three dots indicate mono-, di-, and tri-glycosylated polypeptides. Closed and open arrowheads indicate membrane-protected N-domain and C-domain, respectively. An asterisk indicates glycosylated ProK-resistant core fragment derived from C-domain. (C) Immunoprecipitation of proteinase K fragments with site-specific antibodies. After the proteinase K treatment in the presence (+) or absence (−) of the detergent, peptide fragments were immunoprecipitated with anti–N-domain (IP, N) or anti–C-domain (IP, C) antibody. The total (IP, −) and the immunoprecipitated polypeptides were subjected for SDS-PAGE.

Proline residues between the N-domain and the H-region are critical for SA-I topogenesis. (A) Proline residues were mutated into alanine residues as indicated. (B) The mutants were expressed in a cell-free system in the presence of RM. The upward and downward arrowheads indicate the unglycosylated and glycosylated polypeptides, respectively. In the right panel, the glycosylation efficiency of each construct was determined as described in the legend to Fig. 2. (C) The wild-type and AAA mutant were expressed in the cultured cells. The cells were pulse-labeled and Syt II molecules were immunoprecipitated. Aliquots were further treated by EndoH. (D) Membrane anchoring of the AAA mutant. RM was added during translation reaction (Co) or after the termination of the translation with puromycin (Post). In the latter case (Post), the reaction was continued for a further 30 min at 30°C. The translation products were extracted with 0.1 M Na₂CO₃, and then membrane-bound (M) and soluble (S) fractions were separated by the alkaline floatation procedure. Half equivalents of the total reactions are also shown (T/2). (E) Membrane targeting of AAA mutant of 125 residues. The truncated mRNAs encoding 105 and 125 residues of the AAA mutant were translated in the presence of RM and separated into membrane-bound (P) and supernatant (S) fractions under the high-salt condition. (F) Nascent polypeptide of 125 residues of AAA mutant was cross-linked with Sec61α. After each of the truncated mRNA was translated in the presence of RM, cross-linking (MBS) and immunoprecipitation (IP) were performed as in the legend to Fig. 2.

Working model for topogenesis of the Syt II molecule. Syt-105 is not targeted to the membrane (stage a). Syt-120 and Syt-125 are targeted to the membrane but are not sufficient in length for N-domain translocation (stage b). These are efficiently chased with puromycin to be glycosylated. For completion of the N-domain translocation, further chain elongation is essential so that the positively charged residues beyond the H-region emerge from the ribosome (stage c). Proline residues between the N-domain and the H-region are critical between stage b and stage d to break continuous helix. The threshold of the length for correct integration is 127 residues. The numbers indicate the numbers of amino acid residues of the nascent polypeptide chains.

Timing of cotranslational membrane targeting and N-domain translocation. (A) Template DNAs linearized at various sites were transcribed in vitro. The synthesized mRNAs do not possess a termination codon so that the nascent polypeptides remain as peptidyl-tRNA and are not released from the ribosomes. The polypeptide segments within the ribosomes are indicated by closed ovals. The numbers in the names of the constructs correspond to their length. The hatched box indicates the H-region. (B) Glycosylation of the truncated polypeptides in vitro. The mRNAs were translated in vitro in the absence (−) or presence (+) of RM. The translation reaction was terminated by cycloheximide (C) or puromycin (P). Throughout all of the figures, upward and downward arrowheads indicate the unglycosylated and glycosylated polypeptides, respectively. (C) Effect of puromycin on N-domain translocation. After treatment with either cycloheximide (CHX) or puromycin (Puro), glycosylated and unglycosylated forms were quantified by image analysis and the efficiency (%) was calculated by the formula: [glycosylated-form] × 100/[glycosylated-form + unglycosylated forms]. The experiments for each construct were carried out more than three times and the standard deviation is indicated by error bars. (D) Titration of glycosylation efficiency as a function of polypeptide length. The threshold length for glycosylation is estimated to be 127 residues. (E) Syt-125 is bound to membrane in a high-salt–resistant fashion. Translation mixtures were adjusted to a high-salt condition, and then membrane precipitates (P) and supernatants (S) were separated by ultracentrifugation. The total translation products before separation are also shown (T). Where indicated (Puro), polypeptides were released by puromycin from ribosomes before high-salt treatment. (F) Syt-125 is cross-linked with Sec61α. Translation mixtures were treated with heterobifunctional cross-linker (MBS +). After the reactions, aliquots were immunoprecipitated with anti-Sec61α antibody (IP +). Dot indicates the cross-linked polypeptide (lane 5).

Structural requirements of the H-region for SA-I topogenesis. (A) Effect of serial deletions of the H-region. The glycosylation efficiencies were determined as described in the legend to Fig. 2. (B) The effect of shuffling of the H-region of Syt II with that of the rat cytochrome P450(2C11). The sequences of cytochrome P450(2C11) are underlined.

Effect of partial deletions of the N-domain and replacement of the third proline on N-domain translocation of the AAA mutant. (A) N-domain of the proline mutant (AAA) was partially deleted as indicated. The mutated proline residues and deleted amino acid residues are indicated by a bold letter and a dashed line, respectively. The sequence predicted to form helix is indicated by double underlining. The mutants were expressed in a cell-free system in the presence (+) or absence (−) of RM. The upward and downward arrowheads indicate the unglycosylated and glycosylated polypeptides, respectively. Glycosylation efficiencies were determined as described in the legend to Fig. 2. (B) Prediction of secondary structure of the boundary between N-domain and H-region. H, S, and T correspond to the regions predicted as helix, sheet, and turn, respectively. H-region is indicated by underlines. (C) The third proline residue was exchanged with amino acids with various helical propensities. The glycosylation efficiency was determined as described in the legend to Fig. 2.