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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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RTA binds to ER microsomal membranes. 0.25 μm (A) 2.5 μm (B) RTA259-NBD were incubated in buffer H for 30 min on ice (0 °C) or at 26 °C with either microsomal membranes (+KRM; 20-40 eq) or an equal amount of buffer without microsomes (-KRM). Free RTA259-NBD was then separated from KRM-bound RTA259-NBD either by sedimentation (A) or by gel filtration chromatography (B). A, following sedimentation, the supernatant (s) and the microsomal pellet (p) were analyzed by SDS-PAGE. NBD-labeled proteins were visualized using a fluorescence imager. B, following mixing, free and KRM-bound RTA259-NBD were separated by gel filtration chromatography in buffer H at 4 °C using a Sepharose CL-2B column (8 × 0.5-cm inner diameter). Each fraction was scanned for the presence of RTA259-NBD (•; λex = 468 nm; λem = 530 nm) and KRMs (▾; λex = 405 nm; λem = 420 nm). As controls, only RTA259-NBD (○) or only KRMs (▿) were run and analyzed separately.
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fig1: RTA binds to ER microsomal membranes. 0.25 μm (A) 2.5 μm (B) RTA259-NBD were incubated in buffer H for 30 min on ice (0 °C) or at 26 °C with either microsomal membranes (+KRM; 20-40 eq) or an equal amount of buffer without microsomes (-KRM). Free RTA259-NBD was then separated from KRM-bound RTA259-NBD either by sedimentation (A) or by gel filtration chromatography (B). A, following sedimentation, the supernatant (s) and the microsomal pellet (p) were analyzed by SDS-PAGE. NBD-labeled proteins were visualized using a fluorescence imager. B, following mixing, free and KRM-bound RTA259-NBD were separated by gel filtration chromatography in buffer H at 4 °C using a Sepharose CL-2B column (8 × 0.5-cm inner diameter). Each fraction was scanned for the presence of RTA259-NBD (•; λex = 468 nm; λem = 530 nm) and KRMs (▾; λex = 405 nm; λem = 420 nm). As controls, only RTA259-NBD (○) or only KRMs (▿) were run and analyzed separately.

Mentions: RTA259-NBD Binds to Microsomal ER Membranes—Day et al. (29) showed that RTA binds to liposomes containing negatively charged phospholipids, but RTA binding to natural membranes has not been examined. We therefore investigated whether RTA binds to purified KRMs. Although ricin is exposed first to the lumenal leaflet of the ER membrane after endocytosis, we chose to examine the binding of RTA to the outer surface of ER microsomes because the cytoplasmic leaflet is always exposed in the sealed vesicles. This approach allowed us to focus on RTA with genuine bilayer lipids (including cytoplasmically exposed phosphatidylserine) in the absence of lumenal proteins that might interact with or intercept RTA to complicate interpretation. After incubation of RTA259-NBD with KRMs at 0 or 26 °C, membrane-bound RTA was separated from free protein either by sedimentation or by gel filtration. KRM-exposed RTA259-NBD was found in the pellet following ultracentrifugation, whereas RTA259-NBD remained in the supernatant in the absence of membranes (Fig. 1A). Also, when a sample of RTA259-NBD and KRMs was analyzed by gel filtration, the RTA co-eluted with KRMs in the void volume of the column, whereas KRM-free RTA259-NBD eluted in later fractions (Fig. 1B). Thus, both sedimentation and gel filtration assays demonstrated that RTA259-NBD binds to microsomal membranes with significant affinity. Moreover, this binding occurs at both 0 and 26 °C.


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)

RTA binds to ER microsomal membranes. 0.25 μm (A) 2.5 μm (B) RTA259-NBD were incubated in buffer H for 30 min on ice (0 °C) or at 26 °C with either microsomal membranes (+KRM; 20-40 eq) or an equal amount of buffer without microsomes (-KRM). Free RTA259-NBD was then separated from KRM-bound RTA259-NBD either by sedimentation (A) or by gel filtration chromatography (B). A, following sedimentation, the supernatant (s) and the microsomal pellet (p) were analyzed by SDS-PAGE. NBD-labeled proteins were visualized using a fluorescence imager. B, following mixing, free and KRM-bound RTA259-NBD were separated by gel filtration chromatography in buffer H at 4 °C using a Sepharose CL-2B column (8 × 0.5-cm inner diameter). Each fraction was scanned for the presence of RTA259-NBD (•; λex = 468 nm; λem = 530 nm) and KRMs (▾; λex = 405 nm; λem = 420 nm). As controls, only RTA259-NBD (○) or only KRMs (▿) were run and analyzed separately.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: RTA binds to ER microsomal membranes. 0.25 μm (A) 2.5 μm (B) RTA259-NBD were incubated in buffer H for 30 min on ice (0 °C) or at 26 °C with either microsomal membranes (+KRM; 20-40 eq) or an equal amount of buffer without microsomes (-KRM). Free RTA259-NBD was then separated from KRM-bound RTA259-NBD either by sedimentation (A) or by gel filtration chromatography (B). A, following sedimentation, the supernatant (s) and the microsomal pellet (p) were analyzed by SDS-PAGE. NBD-labeled proteins were visualized using a fluorescence imager. B, following mixing, free and KRM-bound RTA259-NBD were separated by gel filtration chromatography in buffer H at 4 °C using a Sepharose CL-2B column (8 × 0.5-cm inner diameter). Each fraction was scanned for the presence of RTA259-NBD (•; λex = 468 nm; λem = 530 nm) and KRMs (▾; λex = 405 nm; λem = 420 nm). As controls, only RTA259-NBD (○) or only KRMs (▿) were run and analyzed separately.
Mentions: RTA259-NBD Binds to Microsomal ER Membranes—Day et al. (29) showed that RTA binds to liposomes containing negatively charged phospholipids, but RTA binding to natural membranes has not been examined. We therefore investigated whether RTA binds to purified KRMs. Although ricin is exposed first to the lumenal leaflet of the ER membrane after endocytosis, we chose to examine the binding of RTA to the outer surface of ER microsomes because the cytoplasmic leaflet is always exposed in the sealed vesicles. This approach allowed us to focus on RTA with genuine bilayer lipids (including cytoplasmically exposed phosphatidylserine) in the absence of lumenal proteins that might interact with or intercept RTA to complicate interpretation. After incubation of RTA259-NBD with KRMs at 0 or 26 °C, membrane-bound RTA was separated from free protein either by sedimentation or by gel filtration. KRM-exposed RTA259-NBD was found in the pellet following ultracentrifugation, whereas RTA259-NBD remained in the supernatant in the absence of membranes (Fig. 1A). Also, when a sample of RTA259-NBD and KRMs was analyzed by gel filtration, the RTA co-eluted with KRMs in the void volume of the column, whereas KRM-free RTA259-NBD eluted in later fractions (Fig. 1B). Thus, both sedimentation and gel filtration assays demonstrated that RTA259-NBD binds to microsomal membranes with significant affinity. Moreover, this binding occurs at both 0 and 26 °C.

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