ADP‐Ribosylation of Membrane Proteins in Cholinergic Nerve Terminals

Lester, Henry A.; Steer, Michael L.; Michaelson, Daniel M.

doi:10.1111/j.1471-4159.1982.tb05351.x

Cited by 16 publications

(5 citation statements)

References 32 publications

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“…The temperature sensitivity of axonal (Table 1), and t = 2 hr, we find thatj = 1.84 mm/hr. transport, as indicated by its Q1o, has been reported to be [2][3] in several different animals (20,23). If this range is applicable to squid, the rate of fast axonal transport at 37TC can be extrapolated to 190-440 mm/day, which agrees with the rate offast anterograde axonal transport measured in vertebrates, 200-400 mm/day at 370C (20).…”

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confidence: 73%

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Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

Vogel

Chin

Schwartz

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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We rind that half of the pertussis toxinsensitive guanine nucleotide-binding protein (G protein) in the squid (Loligo pealei) giant axon is cytoplasmic and that this species of G protein is intermediate in size between the two forms present in axolemma. This G protein is transported toward synaptic terminals at 44 mm/day. Moreover, these data are consistent with there being two additional steps leading to the maturation of G proteins: (i). association with and transport on intracellular organelles and (ii) modification at the time of transfer to the plasmalemma resulting in a molecular weight shift. Since the other two components of G proteinmediated signal transduction pathways, receptors and effector enzymes, are known to be delivered to the synaptic terminals by fast axonal transport, our rindings introduce the possibility that these three macromolecules are assembled as a complex in the cell body and delivered together to the plasma membrane of the axon and synaptic terminals.G proteins are heterotrimeric, membrane-associated proteins that couple plasma membrane receptors to effector enzymes (1). In nerve cells these signal transduction elements are found in synaptic terminals and growth cones (2-5). The receptors and effectors are integral membrane proteins and, thus, are synthesized in the rough endoplasmic reticulum and then transported to terminals on vesicles by fast axonal transport (6-10). G protein mRNAs do not encode hydrophobic membrane-spanning segments (11), however, so it is unclear when they become associated with membranes and how they are translocated to the plasma membrane. Nonetheless, the subclass of G proteins sensitive to pertussis toxin (PTX) must be transported by synaptic terminals because they, and the receptors that they are known to be coupled with, are located there. If G proteins are indeed carried on cytoplasmic transport vesicles, we should be able to detect them in the axoplasm as they travel from the cell body to the nerve terminal. The approach that we have used to determine whether PTX-sensitive G proteins are associated with transport vesicles is to block axonal transport by focally cooling the axon. When we focally cool (cold block) a short segment of the squid giant axon to 40C, vesicles being transported in the anterograde direction accumulate on the proximal side of the cold block, whereas retrogradely transported vesicles build up on the distal side (12). Segments of axon from both sides of the cooled axon segment can be isolated and assayed for G proteins using PTX-catalyzed ADP-ribosylation and immunoblotting with G protein-specific antibodies. G proteins sensitive to PTX constitute a subclass known to interact with receptors and effectors that are important in controlling neuronal function (13). Tris HCl, pH 7.5/150 mM NaCl/3 mM EDTA/1 mM EGTA, 5 mM 2-mercaptoethanol/5 mM benzamidine) for every centimeter of axon. Extrusion of axoplasm and the recovery of axoplasm and axon sheath (axolemma) were performed by the method described by Lasek (15). Axoplasmic h...

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confidence: 73%

“…In nerve cells these signal transduction elements are found in synaptic terminals and growth cones (2)(3)(4)(5). The receptors and effectors are integral membrane proteins and, thus, are synthesized in the rough endoplasmic reticulum and then transported to terminals on vesicles by fast axonal transport (6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

Vogel

Chin

Schwartz

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous work has provided evidence that G proteins are associated with membranes (Stemweis and Robishaw, 1984;Vogel et al, 1989). They have been localized immunocytochemically in rat brain neuropil (Worley et al, 1986), and the presence ofa G,-like molecule (cholera toxin substrate) in Torpedo synaptosomes has been reported (Lester et al, 1982).…”

Section: Discussionmentioning

confidence: 97%

Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions

et al. 1989

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We prepared and characterized subcellular membrane fractions from the CNS of Aplysia californica that are enriched in isolated nerve terminals (synaptosomes). Ganglia were homogenized in 1.1 M sucrose and fractionated on a 2-step sucrose gradient, yielding 50 micrograms protein/animal in the synaptosomal fraction (P3), which was enriched 3-fold in plasma membrane as compared with the initial homogenate. Quantitative morphometry of electron micrographs revealed that P3 contained 25% intact synaptosomes, a 5-fold enrichment over the homogenate. Although fractionation on a 5-step sucrose gradient reduced the yield of protein in the synaptosomal fraction to 40 micrograms/animal, this fraction (the 0.35 M/0.75 M interface) was more enriched in plasma membrane than P3 and was less contaminated by lysosomes and free mitochondria. By electron microscopy, the 0.35 M/0.75 M interface contained up to 50% synaptosomes. Synaptosomal fractions contained cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinase activities and were enriched in a Mr 40,000 pertussis toxin substrate, Gi/o. In the accompanying paper, we show that these synaptosomes retain the ability to release transmitters.

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“…Taking advantage of the observation that G protein can be covalently labeled by ribosyl ADP in a reaction catalyzed by cholera toxin in other animals (Cassel and Pfeuffer 1978;Gill and Meren 1978;Moss and Vaughn 1979) and the toxin. One prominent component with a molecular weight of 55,000 is labeled, which is a promising candidate for the G protein (Hudson and Johnson 1980;Northrop et al 1980;Lester et al 1982) (Fig. 6).…”

Section: Biochemical Approaches To the Adenylate Cyclase Of Sensory Nmentioning

confidence: 98%

What Molecular Steps Determine the Time Course of the Memory for Short-term Sensitization in Aplysia?

Jh¹,

Bernier²,

Vf³

et al. 1983

Cold Spring Harbor Symposia on Quantitative Biology

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ADP‐Ribosylation of Membrane Proteins in Cholinergic Nerve Terminals

Cited by 16 publications

References 32 publications

Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions

What Molecular Steps Determine the Time Course of the Memory for Short-term Sensitization in Aplysia?

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