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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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Some membrane-bound RTA is stably embedded in the bilayer. RTA259-NBD (500 nm) was incubated with either 40 eq of KRMs (A and D), or 2.5 mm PCPS liposomes (B) in buffer H for 30 min at 20 °C or 37 °C. Membrane-bound RTA was purified by centrifugation and then extracted with alkaline sodium carbonate. The protein contents of the supernatant (s), the membrane pellet (p) fractions, and a molecular weight standard (st) were then analyzed by SDS-PAGE. C, RTA259-NBD (1 μm) was incubated with 5 mm PC liposomes in 10 mm HEPES (pH 7.5) for 30 min at 20 °C or 37 °C. The samples were then treated as in B. D, KRMs were incubated with RTA259-NBD, treated with carbonate, and then purified using a sucrose step gradient as described under “Experimental Procedures.” NBD-labeled proteins were visualized and quantified using a fluorescence imager. Representative gels from a set of at least three independent experiments are shown. Histograms show the average fraction of the total protein in the supernatant and membrane pellet fractions, respectively. The error bars indicate the S.D. of the experiments.
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fig6: Some membrane-bound RTA is stably embedded in the bilayer. RTA259-NBD (500 nm) was incubated with either 40 eq of KRMs (A and D), or 2.5 mm PCPS liposomes (B) in buffer H for 30 min at 20 °C or 37 °C. Membrane-bound RTA was purified by centrifugation and then extracted with alkaline sodium carbonate. The protein contents of the supernatant (s), the membrane pellet (p) fractions, and a molecular weight standard (st) were then analyzed by SDS-PAGE. C, RTA259-NBD (1 μm) was incubated with 5 mm PC liposomes in 10 mm HEPES (pH 7.5) for 30 min at 20 °C or 37 °C. The samples were then treated as in B. D, KRMs were incubated with RTA259-NBD, treated with carbonate, and then purified using a sucrose step gradient as described under “Experimental Procedures.” NBD-labeled proteins were visualized and quantified using a fluorescence imager. Representative gels from a set of at least three independent experiments are shown. Histograms show the average fraction of the total protein in the supernatant and membrane pellet fractions, respectively. The error bars indicate the S.D. of the experiments.

Mentions: Membrane-bound RTA259-NBD Is Resistant to Alkaline Extraction—To determine whether or not RTA is stably embedded in the bilayer core or is bound peripherally to the membrane surface, we examined whether RTA became resistant to alkaline carbonate extraction following its incubation with membranes at various temperatures. After RTA259-NBD was preincubated with KRMs or PCPS liposomes at either 20 or 37 °C, membrane-bound RTA was separated from free protein by sedimentation through a sucrose cushion, and the membranes were treated with sodium carbonate (pH 11.5). The majority of RTA259-NBD incubated at 37 °C was retained in the membrane pellet fraction (KRMs: 71 ± 7%; liposomes: 70 ± 6%; Fig. 6, A, B, and D), thereby indicating that RTA259-NBD was stably integrated into the bilayer. In contrast, at 20 °C, much less RTA259-NBD was found in the carbonate pellet (KRMs: 20 ± 6%; liposomes: 21 ± 9%), suggesting that most membrane-bound RTA was carbonate-extractable (Fig. 6, A, B, and D).


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)

Some membrane-bound RTA is stably embedded in the bilayer. RTA259-NBD (500 nm) was incubated with either 40 eq of KRMs (A and D), or 2.5 mm PCPS liposomes (B) in buffer H for 30 min at 20 °C or 37 °C. Membrane-bound RTA was purified by centrifugation and then extracted with alkaline sodium carbonate. The protein contents of the supernatant (s), the membrane pellet (p) fractions, and a molecular weight standard (st) were then analyzed by SDS-PAGE. C, RTA259-NBD (1 μm) was incubated with 5 mm PC liposomes in 10 mm HEPES (pH 7.5) for 30 min at 20 °C or 37 °C. The samples were then treated as in B. D, KRMs were incubated with RTA259-NBD, treated with carbonate, and then purified using a sucrose step gradient as described under “Experimental Procedures.” NBD-labeled proteins were visualized and quantified using a fluorescence imager. Representative gels from a set of at least three independent experiments are shown. Histograms show the average fraction of the total protein in the supernatant and membrane pellet fractions, respectively. The error bars indicate the S.D. of the experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Some membrane-bound RTA is stably embedded in the bilayer. RTA259-NBD (500 nm) was incubated with either 40 eq of KRMs (A and D), or 2.5 mm PCPS liposomes (B) in buffer H for 30 min at 20 °C or 37 °C. Membrane-bound RTA was purified by centrifugation and then extracted with alkaline sodium carbonate. The protein contents of the supernatant (s), the membrane pellet (p) fractions, and a molecular weight standard (st) were then analyzed by SDS-PAGE. C, RTA259-NBD (1 μm) was incubated with 5 mm PC liposomes in 10 mm HEPES (pH 7.5) for 30 min at 20 °C or 37 °C. The samples were then treated as in B. D, KRMs were incubated with RTA259-NBD, treated with carbonate, and then purified using a sucrose step gradient as described under “Experimental Procedures.” NBD-labeled proteins were visualized and quantified using a fluorescence imager. Representative gels from a set of at least three independent experiments are shown. Histograms show the average fraction of the total protein in the supernatant and membrane pellet fractions, respectively. The error bars indicate the S.D. of the experiments.
Mentions: Membrane-bound RTA259-NBD Is Resistant to Alkaline Extraction—To determine whether or not RTA is stably embedded in the bilayer core or is bound peripherally to the membrane surface, we examined whether RTA became resistant to alkaline carbonate extraction following its incubation with membranes at various temperatures. After RTA259-NBD was preincubated with KRMs or PCPS liposomes at either 20 or 37 °C, membrane-bound RTA was separated from free protein by sedimentation through a sucrose cushion, and the membranes were treated with sodium carbonate (pH 11.5). The majority of RTA259-NBD incubated at 37 °C was retained in the membrane pellet fraction (KRMs: 71 ± 7%; liposomes: 70 ± 6%; Fig. 6, A, B, and D), thereby indicating that RTA259-NBD was stably integrated into the bilayer. In contrast, at 20 °C, much less RTA259-NBD was found in the carbonate pellet (KRMs: 20 ± 6%; liposomes: 21 ± 9%), suggesting that most membrane-bound RTA was carbonate-extractable (Fig. 6, A, B, and D).

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