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Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.

Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, Lord JM, Johnson AE, Roberts LM - J. Biol. Chem. (2009)

Bottom Line: At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer.RTA is then stably bound to the membrane because it is nonextractable with carbonate.Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843-1114, USA.

ABSTRACT
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

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Exposure of RTA259-NBD to the membrane interior. The emission intensity of RTA259-NBD (450 nm in buffer H) was measured before and after the addition of either KRMs (20 eq; A) or 600 μm PCPS liposomes (B). Emission intensities of parallel samples containing either 5NOPC (F5NO) or an equal mol% of POPC (F0) were compared. The net emission intensities are shown as a function of the mol% of 5NOPC in the liposomes (B), but the final mol% of 5NOPC/POPC in the bulk lipid of natural ER membranes (A) cannot be quantified. The averages of at least three independent experiments are shown, and the errorbars indicate the S.D. of the experiments. *, p = 0.208; **, p = 0.006 compared with the quenching efficiency at 20 °C, respectively (Student's t test).
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fig5: Exposure of RTA259-NBD to the membrane interior. The emission intensity of RTA259-NBD (450 nm in buffer H) was measured before and after the addition of either KRMs (20 eq; A) or 600 μm PCPS liposomes (B). Emission intensities of parallel samples containing either 5NOPC (F5NO) or an equal mol% of POPC (F0) were compared. The net emission intensities are shown as a function of the mol% of 5NOPC in the liposomes (B), but the final mol% of 5NOPC/POPC in the bulk lipid of natural ER membranes (A) cannot be quantified. The averages of at least three independent experiments are shown, and the errorbars indicate the S.D. of the experiments. *, p = 0.208; **, p = 0.006 compared with the quenching efficiency at 20 °C, respectively (Student's t test).

Mentions: Upon binding to KRMs containing 5NOPC at 37 °C, RTA259-NBD emission intensity was quenched by the membrane-integrated nitroxide moiety (Fig. 5A). Thus, the dye attached to 259 is exposed to the membrane interior when RTA binds to the microsomal membrane at 37 °C. When incubated at 30 °C, the quenching was much less efficient, and almost no quenching of RTA259-NBD was detected at 20 °C. Because we could not quantify the concentration of 5NOPC within the KRMs, we next prepared PCPS liposomes containing increasing concentrations of 5NOPC. Concentration-dependent quenching curves for the liposomes containing 5NOPC are shown in Fig. 5B. Thus, RTA appears to bind similarly to both KRMs and PCPS liposomes. At 37 °C, maximal quenching efficiency was achieved with liposomes containing about 22.5 mol% 5NOPC. But at the same 5NOPC concentration, quenching was less efficient at 30 °C and was almost undetectable at 20 °C. These data therefore strongly indicate that exposure of the NBD of RTA259-NBD to the nonpolar lipid core of ER microsomes and of PCPS vesicles is strongly temperature-dependent. RTA does not bind to PC liposomes (Fig. 4, C and D), and as expected, no quenching was observed with liposomes containing only POPC and 5NOPC at either 20 °C (F5NO/F0 = 1.0 ± 0.1) or 37 °C (F5NO/F0 = 1.1 ± 0.1).


Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.

Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, Lord JM, Johnson AE, Roberts LM - J. Biol. Chem. (2009)

Exposure of RTA259-NBD to the membrane interior. The emission intensity of RTA259-NBD (450 nm in buffer H) was measured before and after the addition of either KRMs (20 eq; A) or 600 μm PCPS liposomes (B). Emission intensities of parallel samples containing either 5NOPC (F5NO) or an equal mol% of POPC (F0) were compared. The net emission intensities are shown as a function of the mol% of 5NOPC in the liposomes (B), but the final mol% of 5NOPC/POPC in the bulk lipid of natural ER membranes (A) cannot be quantified. The averages of at least three independent experiments are shown, and the errorbars indicate the S.D. of the experiments. *, p = 0.208; **, p = 0.006 compared with the quenching efficiency at 20 °C, respectively (Student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665077&req=5

fig5: Exposure of RTA259-NBD to the membrane interior. The emission intensity of RTA259-NBD (450 nm in buffer H) was measured before and after the addition of either KRMs (20 eq; A) or 600 μm PCPS liposomes (B). Emission intensities of parallel samples containing either 5NOPC (F5NO) or an equal mol% of POPC (F0) were compared. The net emission intensities are shown as a function of the mol% of 5NOPC in the liposomes (B), but the final mol% of 5NOPC/POPC in the bulk lipid of natural ER membranes (A) cannot be quantified. The averages of at least three independent experiments are shown, and the errorbars indicate the S.D. of the experiments. *, p = 0.208; **, p = 0.006 compared with the quenching efficiency at 20 °C, respectively (Student's t test).
Mentions: Upon binding to KRMs containing 5NOPC at 37 °C, RTA259-NBD emission intensity was quenched by the membrane-integrated nitroxide moiety (Fig. 5A). Thus, the dye attached to 259 is exposed to the membrane interior when RTA binds to the microsomal membrane at 37 °C. When incubated at 30 °C, the quenching was much less efficient, and almost no quenching of RTA259-NBD was detected at 20 °C. Because we could not quantify the concentration of 5NOPC within the KRMs, we next prepared PCPS liposomes containing increasing concentrations of 5NOPC. Concentration-dependent quenching curves for the liposomes containing 5NOPC are shown in Fig. 5B. Thus, RTA appears to bind similarly to both KRMs and PCPS liposomes. At 37 °C, maximal quenching efficiency was achieved with liposomes containing about 22.5 mol% 5NOPC. But at the same 5NOPC concentration, quenching was less efficient at 30 °C and was almost undetectable at 20 °C. These data therefore strongly indicate that exposure of the NBD of RTA259-NBD to the nonpolar lipid core of ER microsomes and of PCPS vesicles is strongly temperature-dependent. RTA does not bind to PC liposomes (Fig. 4, C and D), and as expected, no quenching was observed with liposomes containing only POPC and 5NOPC at either 20 °C (F5NO/F0 = 1.0 ± 0.1) or 37 °C (F5NO/F0 = 1.1 ± 0.1).

Bottom Line: At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer.RTA is then stably bound to the membrane because it is nonextractable with carbonate.Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843-1114, USA.

ABSTRACT
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

Show MeSH
Related in: MedlinePlus