Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar 27;23(3):100889.
doi: 10.1016/j.isci.2020.100889. Epub 2020 Feb 7.

A Plant-Specific N-terminal Extension Reveals Evolutionary Functional Divergence Within Translocator Proteins

Affiliations
Free PMC article

A Plant-Specific N-terminal Extension Reveals Evolutionary Functional Divergence Within Translocator Proteins

Pawel Jurkiewicz et al. iScience. .
Free PMC article

Abstract

Conserved translocator proteins (TSPOs) mediate cell stress responses possibly in a cell-type-specific manner. This work reports on the molecular function of plant TSPO and their possible evolutionary divergence. Arabidopsis thaliana TSPO (AtTSPO) is stress induced and has a conserved polybasic, plant-specific N-terminal extension. AtTSPO reduces water loss by depleting aquaporin PIP2;7 in the plasma membrane. Herein, AtTSPO was found to bind phosphoinositides in vitro, but only full-length AtTSPO or chimeric mouse TSPO with an AtTSPO N-terminus bound PI(4,5)P2in vitro and modified PIP2;7 levels in vivo. Expression of AtTSPO but not its N-terminally truncated variant enhanced phospholipase C activity and depleted PI(4,5)P2 from the plasma membrane and its enrichment in Golgi membranes. Deletion or point mutations within the AtTSPO N-terminus affected PI(4,5)P2 binding and almost prevented AtTSPO-PIP2;7 interaction in vivo. The findings imply functional divergence of plant TSPOs from bacterial and animal counterparts via evolutionary acquisition of the phospholipid-interacting N-terminus.

Keywords: Cell Biology; Plant Biology; Plant Physiology.

Conflict of interest statement

Declaration of Interests The authors have no conflict of interest to declare.

Figures

None
Figure 1
Figure 1
Arabidopsis Plants Expressing N-terminally Truncated AtTSPO Are More Affected by Water Loss than Plants Overexpressing Full-Length AtTSPO (A) Percentage water loss after 120 min from soil-grown 17-day-old Arabidopsis (whole aerial parts) grown in a phytotron (average temperature 20°C, approx. 65% humidity, 16 h photoperiod, ∼120 μmol.m−2.s−1) and dehydrated under light in the same conditions. WT, wild-type; KO, transfer DNA insertional AtTSPO knockout line; p35S::AtTSPO, AtTSPO-overexpressing line; p35S::AtTSPOH91A, transgenic line stably overexpressing AtTSPO harboring point mutation H91A; p35S::Venus-AtTSPOΔN, line overexpressing Venus-tagged N-terminally truncated AtTSPO. Pooled measurements from five rosettes coming from at least two pots (distributed randomly in the phytotron) are shown, and red horizontal lines define the means for each dataset. Statistical significance was assessed by one-way ANOVA followed by Tukey's tests (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). (B) Western blot of total protein extracts from rosettes assayed in (A). Per genotype, probed were two replicates (independent pots). (C) Stomatal conductance of intact leaves from soil-grown mature plants under phytotron conditions. Genotypes are as in (A). Four plants (2–3 rosette leaves per plant) were tested for each line. Individual measurements are shown and red horizontal lines define the means for each dataset. Statistical analysis was conducted as in (A), with **p < 0.01. (D) Western blot of total protein extracts from leaves assayed in stomatal conductance experiments.
Figure 2
Figure 2
The Plant-specific N-terminal Extension of AtTSPO Is Required for Interaction and Depletion of PIP2;7 In Vivo (A) Schematic representation of AtTSPO genetic constructs prepared for BiFC analysis. Full-length PIP2;7 and AtTSPO served as positive controls. The N-terminally truncated variant starts with methionine at position 42. (B) Representative confocal images of tobacco epidermal cells transiently coexpressing VenusN-PIP2;7 and VenusC-AtTSPO or VenusN-PIP2;7 and VenusC-AtTSPOΔN. Xylosyltransferase-mTurquoise fluorescent chimera (magenta) was imaged as a cell transfection control (Golgi marker) and for signal quantification. Low-magnification images qualitatively demonstrate the occurrence or lack of BiFC (green), and insets of high magnification images show Golgi stacks (arrowheads). Bars = 20 μm and 5 μm for low and high magnification, respectively. Experiments were repeated three times. (C) Western blot of total extracts from infiltrated leaf areas imaged by BiFC. PIP2;7 expression was detected by anti-GFP. Full-length and truncated AtTSPO were detected by anti-FLAG (tag originally cloned between VenusC and AtTSPO). Non-infiltrated leaf area served as a negative control. (D) Signal quantification shows a drastic reduction in BiFC, and hence interaction between PIP2;7 and truncated AtTSPO mutant (gray histogram) compared with full-length AtTSPO (black histogram). Bars represent means +/− SD. Statistical significance was assessed by an independent samples t test using Graphpad Prism (***p < 0.001). (E) Schematic representation of genetic constructs stably expressed in Arabidopsis suspension cells. The function of the AtTSPO N-terminus was assessed using the mouse homolog (mTSPO) as a negative control and carrier protein for the plant N-terminus. Each construct was N-terminally tagged with GFP. (F) PM fractions were extracted from Arabidopsis suspension cells and PIP2;7 was quantified by western blot. PM proton ATPase (PMA) served as a reference protein for signal quantification. Individual measurements (PIP2;7 signal normalized against PMA signal from 6 to 9 replicates from two independent PM preparations) are shown, and the red horizontal lines define the means. Statistical significance was analyzed by t-tests (D) or one-way ANOVA followed by Tukey's tests (**p < 0.01; ***p < 0.001; ns, not significant) using Graphpad Prism.
Figure 3
Figure 3
Purified AtTSPO Binds Defined Anionic Lipids In Vitro and Binding Requires the N-terminus (A) The N-terminal extension of plant TSPO is positively charged, as shown by ClustalW alignment of the first 50 residues in higher plant (monocot and dicot) TSPO sequences. At, Arabidopsis thaliana; Es, Eutrema salsugineum; St, Solanum tuberosum; Ca, Capsicum annuum; Zm, Zea mays; Os, Oryza sativa; Ta; Triticum aestivum; Pt; Populus trichocarpa. Positively charged amino acids are red (neutral pH) and conserved lysine/arginine residues near the transmembrane domain are indicated by gray rectangles. The mouse (Mus musculus, Mm) TSPO lacks the plant conserved N-terminal extension. (B) Initial screening of AtTSPO ligands yielded several candidate anionic lipids. AtTSPO purified from yeast was incubated with spotted lipids in PIP-strip overlay assays (right panel) and detected with anti-AtTSPO antibodies (left panel). (C) The plant-specific N-terminal extension is involved in AtTSPO-anionic lipid interactions in vitro, as shown by lipid-dependent pull-down of AtTSPO. Purified AtTSPO or AtTSPOΔN[IN] were incubated with PA or agarose resin (negative control). Flow-through [FT], wash (last wash W3), and eluted [E] fractions were probed with HisProbe. (D) Ten histidine-tagged and N-terminally truncated AtTSPOs expressed and solubilized from yeast microsomes were incubated with a gradient of phosphatidic acid concentrations and detected with HisProbe. The bottom panel shows control detection of both proteins. (E) Thermophoresis analyses of full-length AtTSPO and its N-terminal peptide titrated against different phosphatidic acid concentrations. Protein samples were labeled with the red fluorescent dye NT-647-NHS, and non-linear fitting of labeled full-length protein (continuous line) yielded a Kd of 2.1 ± 0.4 mM, compared with a Kd of 3.3 ± 0.9 mM for the N-terminal peptide (dotted line). The rectangular insert is a representative fluorescence trace. For AtTSPO shown is the mean ± SD of three experiments and for AtTSPOΔN shown is a recorded value from one experiment.
Figure 4
Figure 4
The AtTSPO N-terminal Peptide Binds PI(4,5)P2 (A) N-terminally truncated AtTSPO cannot bind PI(4,5)P2in vitro. Thermophoresis was performed by titrating full-length and N-terminally deleted TSPO against different PI(4,5)P2 concentrations. Protein samples were labeled at polyhistidine tags by RED-tris-NTA. Thermophoretic movement of labeled TSPO increases (normalized fluorescence decreases) upon binding PI(4,5)P2. Phosphatidylcholine served as a negative control lipid. Both datasets (AtTSPO + PI(4,5)P2 and AtTSPOΔN + PI(4,5)P2) were compared using two-way ANOVA followed by Bonferroni multiple comparison post-hoc tests. The threshold lipid concentration yielding statistically significant data (p <0.01) is 1.56 mM (arrow). The non-linear regression-based estimate of the Kd for the AtTSPO-PI(4,5)P2 interaction is 2.3 ± 0.7 mM. The rectangular insert is a representative fluorescence trace. For each sample shown is the mean ± SD of three experiments. (B) Purified mouse TSPO homolog lacking the plant-specific N-terminus does not bind PI(4,5)P2in vitro, unlike the full-length plant protein and isolated N-terminal peptide. Five hundred pmol of phosphoinositide was spotted on a nitrocellulose membrane, incubated with purified proteins and detected with anti-AtTSPO (for isolated N-terminus) or HisProbe (for AtTSPO and mTSPO). A mock control of 2% n-dodecyl-β-D-maltoside (DDM) was included. (C) Chimeric mouse TSPO fused to the AtTSPO N-terminus binds PI(4,5)P2in vitro. Purified proteins were incubated with different PI(4,5)P2 concentrations and detected using anti-AtTSPO antibodies (for isolated N-terminal peptide and N-terminus-fused mTSPO) and HisProbe (for AtTSPO and mTSPO). PI3P was spotted as a positive control for mTSPO binding. DDM (2%) served as a mock control. Experiments in (B) and (C) were performed at least twice.
Figure 5
Figure 5
PI(4,5)P2 Biosensor Was Depleted from the PM and Partially Colocalized with AtTSPO in Golgi Membranes in the Presence of Full-length AtTSPO but Not AtTSPOΔN (A) Representative confocal images of Arabidopsis suspension cells stably coexpressing the PI(4,5)P2biosensor (mCherry-tagged double pleckstrin homology domain from phospholipase C; magenta) and full-length AtTSPO or AtTSPOΔN (GFP-tagged; green). Arrowheads indicate mCherry and GFP signals colocalizing at Golgi membranes. * indicates GFP fluorescence in the vacuole. Bars = 10 μm. (B) Solubilized PM-enriched fractions from cells imaged as in (A) were spotted on a nitrocellulose membrane, and PI(4,5)P2 was detected using monoclonal anti-PI(4,5)P2 antibodies. For signal quantification (ImageJ 1.51 software) we used PM proton ATPase (PMA) as a control. (C) Ten-day-old Arabidopsis seedlings grown on MS agar plates were incubated in liquid MS medium with or without 50 μM ABA for 24 h, and PLC activity was measured in triplicate using an EnzCheck direct phospholipase C assay kit. Statistical significance was analyzed by one-way ANOVA followed by Tukey's test (***p < 0.001; **p < 0.01; *p < 0.05). Data in (B) and (C) are means ± SD from three technical replicates, and all experiments were performed at least twice.
Figure 6
Figure 6
The Recombinant AtTSPO N-terminal Peptide Interacts with PI(4,5)P2 and This Anionic Lipid Is Required for AtTSPO Interaction with the Aquaporin PIP2;7 In Vitro (A) 2D 1H-15N HSQC spectrum (500 MHz, 30°C) of 100 μM AtTSPONter in the absence (blue) and in the presence (red) of 25 mM DMPG/50 mM DHPC bicelles. (B) Secondary Structure Propensity (SSP) score in the absence (blue) and in the presence (red) of DMPG/DHPC bicelles. (C) Selected region in 2D 1H-15N HSQC showing different behaviors of residues upon titration of AtTSPONter with increasing amounts of PI(4,5)P2 (0 mM, black; 0.1 mM, blue; 0.2 mM, maroon; 0.3 mM, cyan; 0.4 mM, orange; 0.6 mM, green; 1 mM, red). Residues R36, Q38, K39, R40, and K48 disappear at the first addition of PI(4,5)P2, whereas residues E25, R26, and K27 show progressive chemical shift perturbations. (D) 1H,15N Chemical Shift Perturbations (CSP) after addition of 1 mM PI(4,5)P2. Residues 34–49 that disappear at the first point of the titration are indicated by gray shading. CSPs were calculated as |Δδ(1H)| + |Δδ(15N)|/10. (E) CSP of E26 (green diamonds), R27 (orange circles), K28 (yellow triangles), and A30 (red squares) as a function of PI(4,5)P2 concentration added. (F) Thermophoresis analyses of YFP-PIP2;7 titrated against different AtTSPO concentrations. PIP2;7 movement was followed by YFP fluorescence. Either 50 μM PI(4,5)P2, phosphatidylcholine (PC, negative control), or no lipid was added. Thermophoretic motion of Venus-PIP2;7 decreases (normalized fluorescence increases) upon binding of AtTSPO in the presence of 50 μM PI(4,5)P2 but not PC, nor in the absence of lipids. The horizontal arrow indicates the bound state plateau region. The rectangular insert is a representative fluorescence trace. For each sample shown is the mean ± SD of three experiments. (G) Data from (F) were normalized and fitted using non-linear regression, yielding a typical sigmoidal binding curve only in the presence of PI(4,5)P2. (H) Statistical comparison of Hill slope values from (G). Low pvalues indicate no similarity in steepness between fitted curves.
Figure 7
Figure 7
Specific Positively Charged Lysine-Arginine Pairs in the N-terminal Region of AtTSPO Mediate Interaction with PIP2;7 In Vivo (A) Representative confocal images of tobacco epidermal cells transiently coexpressing VenusN-PIP2;7 and VenusC-AtTSPO or VenusN-PIP2;7 and VenusC-AtTSPO point mutants. Xylosyltransferase-mTurquoise fluorescent chimera (magenta) served as a cell transfection control (Golgi marker) and for signal quantification. Full-length PIP2;7 and AtTSPO served as a positive control for the BiFC signal. Low-magnification images qualitatively demonstrate the occurrence and distribution of BiFC (green) in the transfected area, and insets of high-magnification images show Golgi stacks (arrowheads). For PIP2;7-AtTSPOK35A/R36A, colocalization of the BiFC signal and Golgi marker was not detected. Bars = 20 μm and 5 μm for low and high magnification, respectively. Experiments were repeated three times. (B) Signal quantification showing a drastic reduction in BIFC between PIP2;7 and AtTSPO mutants compared with full-length AtTSPO. Statistical analysis was based on one-way ANOVA followed by Tukey's tests (***p < 0.001). Bars represent means +/− SD. See transparent methods section for quantification procedure. (C) Hypothetical model of PI(4,5)P2-dependent PIP2;7 downregulation by AtTSPO during stress. Under normal conditions (left), synthesized PiP2;7 is targeted to the PM through the biosynthetic secretory pathway. Under osmotic stress conditions (right), expressed AtTSPO is targeted to the ER and Golgi membranes.AtTSPO stimulates PLC activity, depleting PI(4,5)P2 at the PM and generating DAG and PA that modulate the activity of PP2C phosphatases involved in ABA-dependent reduction of water loss through stomata. Decreased PI(4,5)P2 at the PM prevents recruitment of PIP2;7-containing vesicles. Golgi/ER-localized AtTSPO binds PI(4,5)P2 synthesized de novo in these compartments, inducing structural changes needed for recognition and interaction with PIP2;7 en route to the PM, causing redirection to nascent autophagosomes. AtTSPO cannot directly deplete PI(4,5)P2 from the PM but may form a protein complex that recruits PIP5K to organelle/autophagosome initiation sites. AtTSPO-mediated enrichment of ER/Golgi membranes with PI(4,5)P2 may initiate autophagy. Autophagosomal membranes also recruit PIP5K that generates PI(4,5)P2 and the autophagy regulator Atg14/Barkor that interacts with both the enzyme and PI(4,5)P2. PI(4,5)P2 binding to Atg14 regulates its interaction with the Vps34 complex catalyzing PI3P synthesis at autophagosome initiation sites. The presence of the AtTSPO-PI(4,5)P2-PIP2;7 complex in ER/Golgi membranes and the phagophore containing PI(4,5)P2 and Atg8 may be close enough to allow lipid-protein and protein-protein interactions.

Similar articles

See all similar articles

References

    1. Augustine J.J., Bodziak K.A., Hricik D.E. Use of sirolimus in solid organ transplantation. Drugs. 2007;67:369–391. - PubMed
    1. Baba T., Toth D.J., Sengupta N., Kim Y.J., Balla T. Phosphatidylinositol 4,5bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome lysosome fusion. EMBO J. 2019;38:e102837. - PMC - PubMed
    1. Bae K.R., Shim H.J., Balu D., Kim S.R., Yu S.W. Translocator protein 18 kDa negatively regulates inflammation in microglia. J. Neuroimmune Pharmacol. 2014;9:424–437. - PubMed
    1. Banati R.B., Middleton R.J., Chan R., Hatty C.R., Kam W.W., Quin C., Graeber M.B., Parmar A., Zahra D., Callaghan P. Positron emission tomography and functional characterization of a complete PBR/TSPO knockout. Nat. Commun. 2014;5:5452. - PMC - PubMed
    1. Batoko H., Veljanovski V., Jurkiewicz P. Enigmatic Translocator protein (TSPO) and cellular stress regulation. Trends Biochem. Sci. 2015;40:497–503. - PubMed
Feedback