Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Kelner, Katrina L.; Morita, Kyoji; Rossen, Jonathan S.; Pollard, Harvey B.

doi:10.1073/pnas.83.9.2998

Cited by 35 publications

(16 citation statements)

References 34 publications

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“…As a final control, ATP-biotin was incubated with HeLa cells in the presence of lysates containing kinase activity and no biotin signal was observed (Figure S15A), which assures that biotinylation is independent of cell surface protein labeling. Treatment with APB was accompanied by a modest loss of total protein (Figure 4C, lane 10 and 11, bottom gel) compared with controls (Figure 4C, lane 8 and 9, bottom gel), which is similar to the levels of protein loss observed in previous cell permeability studies, [24] including experiments with the widely used cationic-based permeabilization reagents. [25] To assess APB cytotoxicity, a dose-dependent cell viability assay was performed.…”

supporting

confidence: 83%

A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

Fouda

Pflum

2015

Angew Chem Int Ed

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ATP analogs have been powerful tools in the study of kinase-catalyzed phosphorylation. However, the cell impermeability of ATP analogs has largely limited their use to in vitro lysate-based experiments. Here we report the first cell permeable ATP analog, ATP-polyamine-biotin (APB). APB promoted biotin labeling of kinase substrates in live cells. APB has future application in phosphoprotein purification and analysis. More generally, these studies provide a foundation for development of additional cell permeable ATP analogs for cell signaling research.

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supporting

confidence: 83%

A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

Fouda

Pflum

2015

Angew Chem Int Ed

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“…The data shown in Figure 4 indicate that the profile of proteins released by Brij was very constant as a function of time and that there was no apparent sieving on the basis of size. The similarity of the profiles of proteins leaking from detergentpermeabilized cells has been noted before (Kelner et al, 1986;Kellermayer et al, 1986).…”

Section: Permeability To Fluorescent Dyesmentioning

confidence: 92%

Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells

Ridsdale¹,

Clegg²

1991

Journal Cellular Physiology

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Mouse L929 cells were exposed to the nonionic detergent Brij 58. As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase. In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58. The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not escape immediately. This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij. Data are presented that are most readily interpreted as protein leakage having occurred fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent. The permeabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium. Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent. Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.

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“…Accordingly, Kuczenski et al (20) demonstrated that brain TH associated with membranes is functionally more active than soluble brain TH. On the other hand, in chromaffin granules, the membrane-bound TH appears to be present in a "detergent-labile" association with the granule membrane (21) and exposed to the cytoplasm (19). Additionally, Morita et al (22) demonstrated that this association between TH and granule membranes is reversible and specific and that TH activity can be modulated through its association with the granule surface.…”

Section: Discussionmentioning

confidence: 99%

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

Cartier

Parra

Baust

et al. 2010

Journal of Biological Chemistry

115

103

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Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT 2 ) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT 2 physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT 2 , whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT 1 and with overexpressed VMAT 2 . GST pull-down assays further identified three cytosolic domains of VMAT 2 involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT 2 . Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT 2 -containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT 2 /TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT 2 -mediated transport into vesicles.Monoamines, including dopamine (DA), 3 norepinephrine (NE), and serotonin (5-HT), are neurotransmitters that play major roles in a variety of brain functions, including emotion, reward, cognition, memory, attention, locomotion, and stress control (1-6). In neurons and neuroendocrine cells, monoamines are stored in large dense core vesicles (LDCVs) and small synaptic vesicles (SVs) (7-11) that undergo regulated exocytosis through a complex network of protein-protein interactions (12). Loading of monoamines into LDCVs and SVs of neurons and neuroendocrine cells is mediated by two vesicular monoamine transporter isoforms: VMAT 1 (13) and VMAT 2 (14). These transporters contain 12 putative transmembrane domains with both the N and C termini facing the cytosolic side of the vesicle membrane. VMAT 1 is mostly present in LDCVs of neuroendocrine cells, including chromaffin and PC12 cells, whereas VMAT 2 is primarily expressed by monoaminergic neurons of the central nervous system (15). In midbrain DA neurons, VMAT 2 is sorted to LDCVs and SVs in axon terminals and to LDCVs and tubulo-vesicular structures in the somatodendritic compartment (7)(8)(9)(10)(11)15).It is generally accepted that VMAT 2 transports DA that has been previously synthesized in the cytosolic compartment of the presynaptic terminal (16). DA synthesis requires two enzymatic reactions. First, tyrosine hydroxylase (TH) converts tyrosine into DOP...

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Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Cited by 35 publications

References 34 publications

A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

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