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Results: 5

1.
Fig. 5.

Fig. 5. From: Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site.

Hypothetical model for cotranslational N-glycosylation. (A) The monomeric OT forms a platform with the Sec61 complex (Sec) to receive the ribosomes on the ER membrane. (B) The ribosomes bind to the OT–Sec61 complex, forming a supramolecular complex. This binding occurs mainly at the 60S ribosomal subunit. (C and D) During translocation of a nascent polypeptide (C), OT cotranslationally scans it and then transfers N-glycan to glycosylatable sequences in the polypeptide as shown (D).

Yoichiro Harada, et al. Proc Natl Acad Sci U S A. 2009 April 28;106(17):6945-6949.
2.
Fig. 2.

Fig. 2. From: Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site.

Analysis of the interaction between OT, Sec61, and ribosome by linear sucrose density-gradient centrifugation. OT (3.2 pmol of Stt3-MBP), the Sec61 complex (9.5 pmol of Sec61), or Ova (22 pmol) were incubated with (A–C) or without (D) ribosomes (1.7 pmol) for 30 min on ice in the indicated combinations, followed by a further incubation for 30 min at room temperature. The mixture was centrifuged through a 10–40% linear sucrose density-gradient. After manually fractionating the gradient from top to bottom, a small volume (3% of each fraction) was analyzed without additional concentration by Western blot analysis using anti-MBP, anti-FLAG, or anti-Ova antibodies.

Yoichiro Harada, et al. Proc Natl Acad Sci U S A. 2009 April 28;106(17):6945-6949.
3.
Fig. 3.

Fig. 3. From: Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site.

OT, the Sec61 complex, and ribosomes form a ternary complex. (A) OT (7.2 pmol of Stt3-MBP) was incubated with the Sec61 complex (36 pmol of Sec61) and ribosomes (1.7 pmol), and then the mixture was centrifuged through a 10–40% linear sucrose density-gradient. Fractions were recovered from top to bottom of the gradient and then analyzed for A260 to detect ribosomes. Each fraction (3%) was analyzed by a Western blot using anti-MBP and anti-Sec61 antibodies. (B) Ribosomal fractions containing OT, the Sec61 complex, and ribosomes (fractions 7–9 in A) were pooled, sedimented (OT/Sec/Ribo), and then subjected to the Con A pull-down assay (see SI Text). Ten percent of the bound (B) and unbound (U) to Con A beads, and the input (I) was analyzed by SDS/PAGE/Western blot with anti-MBP, anti-Sec61, and anti-Rpl3 antibodies. The ribosomal fractions containing the Sec61 complex and ribosomes (Sec/Ribo) were prepared essentially the same as with OT/Sec/Ribo, and then subjected to the Con A pull-down assay.

Yoichiro Harada, et al. Proc Natl Acad Sci U S A. 2009 April 28;106(17):6945-6949.
4.
Fig. 4.

Fig. 4. From: Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site.

OT directly binds to the 60S subunit near the nascent polypeptide-exiting site. (A) Negative stain electron micrograph of the OT–ribosome binary complex. (Upper) Shown are 4 raw OT-ribosome particle images. The particles are orientated with the 40S subunit on the left, and OT, as indicated by red arrows, on the bottom. (Lower) B1 shows a class image calculated by averaging 23 similar particles. B2 illustrates the likely orientation of ribosome–OT with respect to the ER membrane, which is shown by 2 dashed parallel lines. C1 is a reprojection of a previously-reported 3D cryoEM map of the yeast ribosome-translocon (19), and C2 shows the membrane orientation of the binary complex. (B–D) OT (3.2 pmol) was incubated with a fixed amount of either 80S ribosomes (B), the 60S (C), or the 40S subunit (D) (3.0 pmol each) and then analyzed by either a 10–40% or a 5–20% linear sucrose density-gradient centrifugation.

Yoichiro Harada, et al. Proc Natl Acad Sci U S A. 2009 April 28;106(17):6945-6949.
5.
Fig. 1.

Fig. 1. From: Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site.

Catalytically-active OT binds to ribosomes. (A) OT, the Sec61 complex, and ribosomes (3.2, 9.5, and 1.7 pmol, respectively) were incubated in the indicated combinations, followed by sedimentation through a 20% sucrose cushion containing 0.1% deoxy Big CHAP at 200,000 × g for 2 h. The pellet (30%) was analyzed by SDS/PAGE followed by Western blot analysis with anti-MBP and anti-Sec61 antibodies. (B) The OT activities in the original OT (n = 3) and the pellet (n = 3) that was sedimented through a 20% sucrose cushion containing 0.125% digitonin and 25 μg/mL phosphatidylcholine at 200,000 × g for 2 h are shown. The pellet fractions were prepared from OT (7.2 pmol of Stt3-MBP), the Sec61 complex (36 pmol of Sec61), and 1.7 pmol of ribosomes in the indicated combinations. The protein amount of OT in the pellet fraction was estimated by Western blot analysis with anti-MBP antibody. The intensity of Stt3-MBP in the purified OT (320 fmol) was compared with that in the pellet fraction. (C) The pellet in B was analyzed by SDS/PAGE followed by silver staining (Right). The purified OT (100 ng), Sec61 (100 ng), and ribosomes (800 ng) were analyzed on the same gel (Left).

Yoichiro Harada, et al. Proc Natl Acad Sci U S A. 2009 April 28;106(17):6945-6949.

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