Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates ...adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.
In this study, we synthesized and evaluated a new spirocyclic piperidine derivative 3, containing a 4‐fluorobutyl side chain, as a PET radioligand for neuroimaging of σ1 receptors. In vitro, compound ...3 displayed high affinity for σ1 receptors (Ki=1.2 nM) as well as high selectivity. 18F3 radiosynthesis was performed from the corresponding tosylate precursor, with high radiochemical yield (45–51 %), purity (>98 %), and specific activity (>201 GBq μmol−1). Metabolic stability of 18F3 in the brain of CD‐1 mice was verified, and no penetration of peripheral radiometabolites into the cerebral tissue was observed. Results of ex vivo autoradiography revealed that the distribution of 18F3 in the brain corresponded to regions with high σ1 receptor density. The highest region‐specific total‐to‐nonspecific ratio was determined in the facial nucleus (4.00). Biodistribution studies indicated rapid and high levels in brain uptake of 18F3 (2.2 % ID per gram at 5 min p.i.). Pre‐administration of haloperidol significantly inhibited 18F3 uptake into the brain and σ1 receptor‐expressing organs, further confirming in vivo target specificity.
PET project for σ1 receptors: A new fluoroalkyl‐substituted spirocyclic piperidine 3 was synthesized and revealed to have high σ1 receptor affinity (Ki=1.2 nM). This derivative was labeled with 18F, and initial in vivo experiments provided evidence for the suitability of 18F3 for PET imaging of σ1 receptors.
The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH ...increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO2, HCl or NH3 differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO2 either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO2 shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO2 was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO2-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO2 under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO2 stress increased in the presence of oxygen with the length of previous exposure to high CO2. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO2 was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO2, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO2 stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m2 leaf area)-1 s-1 or less. Proton export under acid stress into the vacuole was about 1200 nmol m-2s-1. The kinetics of pH responses to the addition or withdrawal of CO2 indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast.
Summary
Transport Protein Particle
II
(
TRAPPII
) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little ...information is known about Arabidopsis
TRAPPII
complex topology and molecular function. In this study, independent proteomic approaches initiated with
TRAPP
components or Rab‐A
GTP
ase variants converge on the
TRAPPII
complex. We show that the Arabidopsis genome encodes the full complement of 13
TRAPPC
subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between
TRAPPII
subunits. Preferential binding to dominant negative (
GDP
‐bound) versus wild‐type or constitutively active (
GTP
‐bound)
RAB
‐A2a variants discriminates between
TRAPPII
and
TRAPPIII
subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan
TRAPP
complexes. Analyzes of Rab‐A mutant variants in
trappii
backgrounds provide genetic evidence that
TRAPPII
functions upstream of
RAB
‐A2a, allowing us to propose that
TRAPPII
is likely to behave as a guanine nucleotide exchange factor (
GEF
) for the
RAB
‐A2a
GTP
ase.
GEF
s catalyze exchange of
GDP
for
GTP
; the
GTP
‐bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding
GEF
−Rab interactions will be crucial to unravel the co‐ordination of plant membrane traffic.
Significance Statement
The Arabidopsis proteome contains two distinct sets of TRAPP subunits, the first of which resembles metazoan TRAPPII, preferentially binds RAB‐A2a, and may act as a Guanosine Exchange Factor (GEF) for the TGN‐associated Rab‐A2 subclade of GTPases. The second set of TRAPP subunits resembles metazoan TRAPPIII and preferentially binds the vacuolar GTPase RAB‐G3f over RAB‐A2a, indicating that TRAPPIII may mediate degradative trafficking routes whilst TRAPPII mediates biosynthetic routes.
In order to develop a fluorinated radiotracer for imaging of σ(1) receptors in the central nervous system a series of (2-fluoroethyl) substituted spirocyclic piperidines 3 has been prepared. In the ...key step of the synthesis 2-bromocinnamaldehyde acetal 5 was added to piperidones 6 with various substituents at the N-atom. Unexpectedly, this reaction led to 2-benzoxepines 8, which were contracted with acid to afford the spirocyclic 2-benzofuranacetaldehydes 9. The best yields were obtained, when the transformations up to the alcohols 10 were performed without isolation of intermediates. Generally the (2-fluoroethyl) derivatives 3 have higher σ(1) affinity and σ(1)/σ(2) selectivity than the corresponding (3-fluoropropyl) derivatives 2. The most promising candidate for the development as radiotracer is the (2-fluoroethyl) derivative 3a (WMS-1828, fluspidine, 1'-benzyl-3-(2-fluoroethyl)-3H-spiro2benzofuran-1,4'-piperidine), which shows subnanomolar σ(1) affinity (K(i)=0.59nM) and excellent selectivity over the σ(2) subtype (1331-fold) as well as some other receptor systems. The novel synthetic strategy also allows the systematic pharmacological evaluation of intermediate alcohols 10. Despite their high σ(1) affinity (K(i)=6-32nM) and selectivity the alcohols 10 are 10-30-fold less potent than the bioisosteric fluoro derivatives 3.
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