Exocyst-Positive Organelles and Autophagosomes Are Distinct Organelles in Plants^,

Exocyst-positive organelles are distinct from autophagosomes under normal growth conditions but overlap in the vacuole after autophagic induction.

Abstract

Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner.

Protein transport between intracellular organelles and the plasma membrane (PM) is mediated by vesicles (Park and Jürgens, 2011; Brandizzi and Barlowe, 2013). However, successful protein trafficking is not only a question of selective sorting and packaging of cargo molecules into vesicles (Schu, 2001; Sato and Nakano, 2007), but is also the result of correct targeting, capture, and recognition by the acceptor membranes (Sztul and Lupashin, 2006). Two types of protein complexes serve this purpose: long-range tethering factors (Yu and Hughson, 2010) and cognate v- and t-soluble NSF attachment protein receptors (SNAREs; Jahn and Scheller, 2006). Many of the tethering factors are multisubunit protein complexes, probably the most well-known example being the exocyst. This is an octameric complex containing the proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84, and was originally described in yeast (Saccharomyces cerevisiae) as a complex mediating the fusion of secretory vesicles with the PM (TerBush et al., 1996). In the meantime, it is now recognized as being ubiquitously required for exocytic events in all eukaryotic cells (Heider and Munson, 2012). The exocyst complex also facilitates a number of other membrane fusion processes such as cytokinesis (Neto and Gould, 2011; Rybak et al., 2014) and autophagy (Bodemann et al., 2011). Plants also have an exocyst complex, and there are a number of papers implicating its role in secretion (Kulich et al., 2010; Li et al., 2013; Safavian and Goring, 2013; Cole et al., 2014). An interesting feature of the plant exocyst complex is the amplification of some of its subunits, in particular Exo70, which in Arabidopsis (Arabidopsis thaliana) has 23 paralogs (Synek et al., 2006). This has led to the suggestion that plants may have multiple exocyst complexes (Cvrčková et al., 2012). Indeed, whereas an Exo70A1-type exocyst is required for conventional secretory processes (Hála et al., 2008; Zárský et al., 2009), an Exo70E2-type exocyst appears to be characteristic for unconventional protein secretion (Wang et al., 2010; Ding et al., 2012, 2014a, 2014b). Furthermore, Exo70B1 seems to be specifically needed for the fusion of autophagosomes with the vacuole (Kulich et al., 2013).

The Exo70E2-positive structures, termed exocyst-positive organelles (EXPO), in plants are 500 to 800 nm in diameter and have two boundary membranes (Wang et al., 2010; Ding et al., 2014b). As such, they are morphologically indistinguishable from autophagosomes, both in animals (Yang and Klionsky, 2010) and in plants (Zhuang et al., 2013). Unlike autophagosomes, which ultimately fuse with the lytic compartment of the cell (lysosome or vacuole), EXPO deliver the luminal contents into the apoplast first by fusion of the outer membrane with the PM, then by degradation of the inner boundary membrane (Wang et al., 2010; Ding et al., 2012). The authenticity of EXPO has been challenged by Kulich et al. (2013), who have claimed that “overexpression of exocyst subunits itself may cause stimulation of biogenesis of new exocyst positive compartments that are normally absent.” Although it cannot be ruled out that overexpression of Exo70E2 may result in an increase in EXPO numbers, indeed overexpression of Arabidopsis Exo70E2 (AtExo70E2) in mammalian cells causes EXPO-like structures to be formed (Ding et al., 2014b), and similar EXPO densities are obtained in Arabidopsis protoplasts when the expression is driven by the Exo70E2 native promoter (Ding et al., 2014b). More importantly, EXPO can be immunologically detected in wild-type cells with Exo70E2 antibodies (Wang et al., 2010). Nevertheless, the structural similarity between EXPO and autophagosomes suggests that they might in some way be related or follow the same biogenetic route in the cell.

In this paper, we describe the fate of EXPO in suspension-cultured cells of Arabidopsis as well as in Arabidopsis root cells after the induction of autophagy. Visualization of EXPO and autophagosomes was done in cells expressing fluorescent AtExo70E2 or Arabidopsis autophagy-related gene8 (AtATG8), a well-known autophagosome marker, or both, as well as immunologically with specific antibodies against Exo70E2 and ATG8. In untreated cells, it is clear that EXPO and autophagosomes are separate and distinct organelles. However, after induction of autophagy in the presence of concanamycin A (ConcA), EXPO signals are lost from the PM and instead are gradually detected, together with AtATG8 signals, in the vacuole lumen. These signals partially overlap with FM4-64, which was used to stain the tonoplast. This points to an internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. Although autophagy caused an increase in the amount of ATG8 recruited to membranes, a similar effect was not observed with AtExo70E2.

RESULTS

Under Normal Growth Conditions, EXPO and Autophagosomes Are Separate and Distinct Organelles

We have performed immunofluorescent staining using Exo70E2 and ATG8e antibodies to localize the endogenous proteins in transgenic Arabidopsis cells expressing either the EXPO marker Exo70E2-GFP (Wang et al., 2010; Zhuang et al., 2013) or the autophagosomal marker yellow fluorescent protein (YFP)-ATG8f (Contento et al., 2005; Phillips et al., 2008; Wang et al., 2011) under the control of the Cauliflower mosaic virus (CaMV) 35S promoter or the ubiquitin 10 promoter, respectively. The specificity of both antisera was first proven by clear recognition of the Exo70E2 or ATG8e antibody (Wang et al., 2010; Zhuang et al., 2013) to either Exo70E2-GFP or YFP-ATG8f/YFP-ATG8e, respectively, via immunofluorescence labeling or the detection of endogenous Exo70E2 or ATG8e proteins by western blotting (Supplemental Fig. S1). The ATG8e antibody was then used to label endogenous ATG8e in the Exo70E2 cell line, whereas the Exo70E2 antibody was utilized to detect the endogenous Exo70E2 level in the ATG8f cell line. Significantly, the cross-labeling results showed that, under normal growth conditions, the fluorescent signals from Exo70E2-GFP or YFP-ATG8f did not colocalize with the fluorescent signals coming from the ATG8e or Exo70E2 antibody, respectively (Pearson correlation coefficient: −0.40 ± 0.06 and −0.45 ± 0.08, respectively; Fig. 1, A and B; Supplemental Fig. S2). This indicates that EXPO are indeed separate compartments from autophagosomes in nonstressed transgenic culture cells (Fig. 1, A and B). This notion was further supported by cross labeling using the transgenic plants expressing either YFP-ATG8e or Exo70E2-GFP (Pearson correlation coefficient: −0.52 ± 0.09 and −0.29 ± 0.15, respectively; Fig. 1, C and D; Supplemental Fig. S2).

Figure 1.

EXPO and autophagosomes are distinct organelles under normal growth conditions. Under normal growth conditions, endogenous ATG8e labeled by ATG8e antibodies was separate from the EXPO marker Exo70E2-GFP in transgenic Arabidopsis cells (A) or plants (C). Conversely, endogenous Exo70E2 labeled by Exo70E2 antibodies was also separate from the autophagosomal marker YFP-ATG8f/e in transgenic Arabidopsis cells (B) or plants (D), respectively. Scale bars = 10 µm.

Upon Autophagic Induction, Exo70E2-GFP and YFP-ATG8f/e Gradually Lose Their Typical Localization and Become Internalized in the Vacuole as Autophagic Bodies

To study the fate of EXPO during autophagy induction, and therefore to test for a possible relationship to autophagosomes, both transgenic cell lines were subjected to autophagic induction by treatment with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH; Yoshimoto et al., 2009; Wang et al., 2011; Zhuang et al., 2013), together with ConcA, a V-ATPase inhibitor that prevents vacuolar acidification (Dettmer et al., 2006). Under autophagic conditions, ConcA therefore prevents the degradation of autophagosomes that have fused with the tonoplast, allowing single-membrane-bound autophagic bodies to accumulate in the vacuole lumen (Klionsky and Ohsumi, 1999; Yoshimoto et al., 2004).

Without autophagic induction, both Exo70E2-GFP and YFP-ATG8f/e exhibited typical and distinct punctate patterns in both Arabidopsis cell lines and transgenic plants. In agreement with its proposed role in exocytosis, Exo70E2-GFP is found mainly adjacent to the PM (Fig. 2, A and C), whereas YFP-ATG8f/YFP-ATG8e tended to produce a diffuse cytosolic fluorescence with few punctae (Fig. 2, B and D; see also Zhuang et al., 2013; Bassham, 2015). With the onset of autophagy and in the presence of ConcA, Exo70E2-GFP gradually began to lose its typical peri-PM punctate pattern, and a diffuse cytosolic signal became visible (Fig. 2, A and C). At later stages of autophagy, Exo70E2 signals were clearly seen within the lumen of the vacuole (Fig. 2, A and C). The autophagosomal marker YFP-ATG8f responded to autophagic induction by forming more cytosolic punctae (indicated as arrows in Fig. 2B) as previously reported (Zhuang et al., 2013). With prolonged autophagy, YFP-ATG8f/YFP-ATG8e signals also accumulated within the vacuole lumen (Fig. 2, B and D). Confocal analysis at different time points confirmed that the numbers of Exo70E2- and ATG8f-positive signals were larger at later stages of autophagic induction (punctate size at the late stage compared to the early stage with either Exo70E2 or ATG8f is about 2.6-fold larger), which may represent their temporary buildup in the cytoplasm before deposition in the vacuole lumen (Zhuang et al., 2013). We then further confirmed these observations with other autophagic induction conditions. After Suc or nitrogen depletion, fluorescent signals from Exo70E2 or ATG8e/8f were also observed in the vacuole lumen in transgenic cell lines or plants (Supplemental Figs. S3 and S4).

Figure 2.

Turnover of EXPO and autophagosomes in response to BTH-induced autophagy. Nonstressed Arabidopsis transgenic cells (A and B) or plants (C and D) expressing either Exo70E2-GFP (A and C) or YFP-ATG8f/e (B and D) showed punctate fluorescent structures representing, respectively, EXPO or autophagosomes. Upon autophagic induction by BTH (100 µm) and ConcA (0.5 µm) treatment (8 h for cells and 5 h for plants; early stage), both Exo70E2-GFP and YFP-ATG8f/e labeled autophagic bodies (arrows) inside vacuoles, and the amount of autophagic bodies greatly increased with time (16-h treatment for cells and 10-h treatment for plants). Scale bars = 10 µm.

Autophagy-Induced Vacuolar Internalization of EXPO Can Also Be Visualized in Transgenic Plants Expressing both YFP-ATG8e and AtExo70E2-mRFP

To rule out overexpression artifacts caused by the use of the 35S CaMV promoter, we generated a transgenic plant expressing AtExo70E2-GFP under its endogenous promoter (Fig. 3A). Although the strength of the Exo70E2-GFP signals in root cap cells was weaker when using the endogenous promoter, there was no obvious difference in signal distribution when compared with the fluorescent signals from AtExo70E2-GFP under the 35S promoter (Fig. 3B), indicating the expression pattern of Exo70E2-GFP previously described (Wang et al., 2010; Ding et al., 2014b) is not the overexpression artifact. Moreover, fluorescent Exo70E2 and ATG8e signals from Arabidopsis transgenic plants expressing both YFP-ATG8e and Exo70E2-mRFP in normal cells remained distinct and separate from one another (Pearson correlation coefficient: −0.30 ± 0.08; Fig. 3C; Supplemental Fig. S5). The presence of overlapped fluorescent Exo70E2 and ATG8e signals in the vacuole was also observed after either BTH treatment or nitrogen starvation plus ConcA treatment (Pearson correlation coefficient: 0.17 ± 0.09 and 0.22 ± 0.03 at early stage, and 0.57 ± 0.09 and 0.56 ± 0.08 at late stage [Fig. 3, D and E, respectively]; Supplemental Fig. S5).

Figure 3.

EXPO and autophagosome marker signals are separate under normal conditions but colocalize after autophagic induction in double transgenic plants. A, Diagram showing the constructs in which expression of AtExo70E2-GFP is driven by either the endogenous Exo70E2 promoter or the 35S CaMV promoter. B, Transgenic Arabidopsis plants expressing AtExo70E2-GFP driven by either the endogenous Exo70E2 promoter or the 35S CaMV promoter showed a typical EXPO punctate pattern (also see Wang et al., 2010; Ding et al., 2014b). C, In transgenic Arabidopsis plants expressing both YFP-ATG8e and AtExo70E2-monomeric red fluorescent protein (mRFP), the different fluorescent signals were distinct from each other under normal conditions. D, Upon autophagic induction by BTH (100 µm) plus ConcA (0.5 µm) treatment, YFP-ATG8e and AtExo70E2-mRFP showed partial colocalization (indicated with the arrow) at early stages (5 h), and the colocalization ratio greatly increased (indicated with four arrows) at late stages (10 h). E, Upon autophagic induction by nitrogen starvation plus ConcA (0.5 µm) treatment, YFP-ATG8e and AtExo70E2-mRFP showed partial colocalization (indicated with the arrow) at early stages (12 h), and the colocalization ratio greatly increased (indicated with five arrows) at late stages (24 h). Scale bar = 10 µm. DIC, Differential interference contrast; Pro, promoter; NOS ter, nopaline synthase terminator.

FM4-64 Staining Confirms Autophagy Induced Vacuolar Internalization of EXPO and Autophagosomes

To discern more precisely the position of the tonoplast, Arabidopsis roots were exposed to the styryl dye FM4-64. This dye is initially taken up into the outer lipid monolayer of the PM and over time is internalized via endocytosis before accumulating in the tonoplast (Bolte et al., 2004; Dettmer et al., 2006). At this stage, few if any punctate fluorescent FM4-64 signals were visible inside the vacuole of normal cells (Fig. 4A; about 1% of total fluorescent signals inside vacuole; Supplemental Fig. S5), and the distribution of Exo70E2-GFP and YFP-ATG8e was as described above (Figs. 1 and 2). However, autophagy induced in the presence of ConcA caused increasing numbers of fluorescent FM4-64 punctae to become visible within the lumen of the vacuoles (Fig. 4, B and C; about 30% or 70% of total fluorescent signals inside the vacuole for either early or late stages, respectively; Supplemental Fig. S5). Some of the FM4-64 punctae colocalized with internalized Exo70E2-GFP and YFP-ATG8e signals (Pearson correlation coefficients are around 0.35 at late stage; Supplemental Fig. S5), but in each case, there was a surplus of noncolocalized FM4-64 punctae (Fig. 4, B and C). Since the fluorescent punctae in the vacuole were quite mobile, and many of the Exo70E2-GFP/YFP-ATG8e were observed to lie closely adjacent, the actual degree of colocalization may have been higher due to an insufficient speed of image acquisition, although wavelength switching was performed as rapidly as our confocal laser scanning microscopy would allow.

Figure 4.

FM4-64 staining improves visualization of Exo70E2-GFP and YFP-ATG8e signals that are internalized into vacuolar lumen due to autophagy. A, Under normal conditions, transgenic Arabidopsis plants expressing either Exo70E2-GFP under 35S CaMV or Exo70E2 native promoters, or YFP-ATG8e showed punctae representing EXPO and autophagosomes, respectively, and EXPO were mainly localized on the plasma membrane. B and C, Transgenic Arabidopsis plants expressing Exo70E2-GFP under either 35S CaMV promoter (B) or different promoters (C), or transgenic Arabidopsis plants expressing YFP-ATG8e (B and C) showed autophagic bodies inside the vacuole after autophagic induction by BTH (100 µm) and ConcA treatment (0.5 µm) for 5 h (early stage) or 10 h (late stage). The tonoplast was clearly labeled by FM4-64. The autophagic bodies (arrows) partially overlapped with internalized FM4-64 punctae. Besides, FM4-64 could also label some surplus punctae inside vacuole lumen (arrowheads). Scale bar = 10 µm.

To rule out that the turnover of Exo70E2 fluorescent signals in the vacuole lumen is an artifact of overexpression, we further tested this phenomenon by using stable transgenic Arabidopsis plants expressing Exo70E2-GFP under the control of the Exo70E2 native promoter as experimental materials (Fig. 4; Supplemental Fig. S5). Under normal conditions, Exo70E2-GFP signals were mainly located at the peripheral of the cells as described above (Fig. 4A). However, Exo70E2-GFP signals translocated into the vacuole lumen and partially colocalizated with FM4-64-labeled punctae at the late stage of autophagic induction (Pearson correlation coefficient is around 0.40 at late stage; Fig. 4C; Supplemental Fig. S5), which further confirmed our observations by using overexpression plants as materials.

The unexpected observation of large numbers of FM4-64-stained punctae in the vacuole lumen after autophagic induction in the presence of ConcA caused us to perform two sets of control experiments. Firstly, we examined whether autophagic conditions or ConcA was responsible. As seen in Figure 5, BTH-induced autophagy alone failed to cause an obvious redistribution of Exo70E2-GFP signals, and even after long periods of autophagic induction (Fig. 5B; Supplemental Fig. S4), little or no Exo70E2-GFP or YFP-ATG8e fluorescence was seen in the vacuole lumen (about 10% of total fluorescent signals inside vacuole; Supplemental Fig. S4). FM4-64-stained punctae in the vacuole lumen were also much reduced (about 20% of total fluorescent signals inside the vacuole; Fig. 5B; Supplemental Fig. S5). In contrast, ConcA treatment alone led to the accumulation of both FM4-64 and Exo70E2-GFP or FM4-64 and YFP-ATG8e signals in the vacuoles of transgenic Arabidopsis root cells (Fig. 5; Supplemental Figs. S4 and S5). Interestingly, FM4-64-stained punctae were detected in the vacuole in the absence of BTH treatment at a time point (5 h) when the great majority of the Exo70E2-GFP labeling was still to be at the cell surface (Fig. 5A). We next asked whether the visualization of FM4-64-stained punctae in the vacuole was a specific effect of the dye itself. We therefore tested two well-known fluorescent tonoplast markers, YFP-VAMP711 (Geldner et al., 2009) and YFP-RABG3f (YFP-RABG3f also labels late endosome; Cui et al., 2014; Ebine et al., 2014; Singh et al., 2014), in conjunction with FM4-64 to stain the tonoplast. As seen in Supplemental Figure S6, fluorescent VAMP711 and RABG3f punctae, with considerable FM4-64 colocalization (Pearson correlation coefficient: 0.41 ± 0.06 and 0.47 ± 0.07, respectively), were seen inside the vacuoles of root cells from both transgenic lines after autophagic induction in the presence of ConcA, and were also observed with ConcA treatment alone, although with less colocalization (Pearson correlation coefficient: 0.20 ± 0.06 and 0.15 ± 0.03, respectively).

Figure 5.

Both Exo70E2-GFP and YFP-ATG8e signals can be observed in the vacuolar lumen after ConcA treatment. A and B, Transgenic Arabidopsis plants expressing Exo70E2-GFP or YFP-ATG8e showed punctae presumably as autophagic bodies inside the vacuole after ConcA treatment (0.5 µm) for 5 h (early stage) or 10 h (late stage). Those punctae cannot be observed after BTH (100 µm) treatment. Conversely, FM4-64 labeled-punctae in vacuole lumen were observed after both BTH and ConcA treatment at different stages. Scale bar = 10 µm.

Due to the possibility that the vacuolar lumen-located fluorescent punctae from either Exo70E2-GFP, YFP-ATG8e, YFP-RABG3f, YFP-VAMP711, or FM4-64 after autophagic induction could be caused by the single-layer imaging of cytoplasmic channels traversing the vacuole or from deep invaginations of the tonoplast, we therefore conducted three-dimensional imaging of different samples (Supplemental Fig. S7; Supplemental Movies S1, S2, S3, and S4). The punctae from plants expressing Exo70E2-GFP, YFP-ATG8e, YFP-RABG3f, YFP-VAMP711, or FM4-64 were only observed in a single layer of the z-stack images, which does not conform with the possibility described above. In addition, three-dimensional reconstruction of z-stack images clearly showed that such punctae are really inside the vacuole (Supplemental Movies S1, S2, S3, and S4).

Exo70E2-GFP (EXPO) and YFP-ATGf/e (Autophagosomes) Gradually Colocalize during Autophagic Induction

Having established that autophagy induces a vacuolar internalization of fluorescent markers for both EXPO and autophagosomes, we next asked whether these structures were internalized separately or together. We first performed transient coexpression of YFP-ATG8e and Exo70E2-mRFP, and found that these two markers were distinct at the early stage after electroporation but gradually showed colocalization at later stages of autophagic induction (Supplemental Fig. S8). To rule out transient recruitment of AtExo70E2 (Ding et al., 2014b), we therefore determined the degrees of colocalization of Exo70E2-GFP with ATG8e antibodies or YFP-ATG8f/YFP-ATG8e with Exo70E2 antibodies after 8 or 5 h (early stage for transgenic cells or transgenic plants, respectively) and 16 or 10 h (late stage for transgenic cells or transgenic plants, respectively) of autophagy induction in stably transformed materials. At the early stage of autophagy, both cytosolic and vacuolar signals for the two markers were visible, and there was only little overlap in the two signals (Fig. 6; Supplemental Fig. S2). However, at a later stage of autophagy when the majority of the Exo70E2-GFP and YFP-ATG8f/YFP-ATG8e signals were present inside the vacuole, a high degree of signal overlap was observed (Fig. 7; Supplemental Fig. S2). To exclude that the colocalization of these two markers is caused by overexpression, we further performed the same experiment with transgenic Arabidopsis plants expressing Exo70E2-GFP under the control of the Exo70E2 native promoter. Once again, Exo70E2-GFP signals were separated from signals from ATG8e antibodies under normal conditions (Fig. 8; Pearson correlation coefficient: −0.46 ± 0.09; Supplemental Fig. S5), but gradually showed colocalization after autophagic induction (Fig. 8; Pearson correlation coefficient: 0.13 ± 0.06 and 0.71 ± 0.05 for early and late stages, respectively; Supplemental Fig. S5).

Figure 6.

Immunofluorescent labeling analysis of the turnover of EXPO and autophagosomes at the early stage of autophagic induction. At the early stage of autophagic induction, Exo70E2-GFP signals partially colocalized with endogenous ATG8e signals as recognized by ATG8e antibodies in transgenic Arabidopsis cells (A) or plants (C). Conversely, YFP-ATG8f (transgenic cells)/YFP-ATG8e (transgenic plants) signals partially colocalized with endogenous Exo70E2 as labeled by Exo70E2 antibodies in transgenic Arabidopsis cells (B) or plants (D). Early stage, 8-h BTH (100 µm) and ConcA (0.5 µm) treatment in transgenic Arabidopsis cells and 5-h BTH and ConcA treatment in transgenic Arabidopsis plants; scale bars = 10 µm.

Figure 7.

Immunofluorescent labeling analysis of the turnover of EXPO and autophagosomes at the late stage of autophagic induction. At the late stage of autophagic induction, Exo70E2-GFP signals colocalized with endogenous ATG8e as recognized by ATG8e antibodies in the vacuolar lumen in transgenic Arabidopsis cells (A) or plants (C). Similarly, YFP-ATG8f (transgenic cells)/YFP-ATG8e (transgenic plants) signals colocalized with endogenous Exo70E2 as recognized by Exo70E2 antibodies in transgenic Arabidopsis cells (B) or plants (D). Late stage, 16-h BTH (100 µm) and ConcA (0.5 µm) treatment (transgenic Arabidopsis cells) and 10-h BTH (100 µm) and ConcA (0.5 µm) treatment (transgenic Arabidopsis plants). Scale bars = 10 µm.

Figure 8.

Exo70E2-GFP signals from transgenic Arabidopsis plants driven by the Exo70E2 native promoter are separate from autophagosomal marker signals under normal conditions but ultimately colocalize after induction of autophagy. A, Under normal growth conditions, Exo70E2-GFP signals are distinct from endogenous ATG8e as recognized by ATG8e antibodies. B, After treatment with BTH (100 µm) plus ConcA (0.5 µm), Exo70E2-GFP signals gradually show a partial colocalization with endogenous ATG8e (recognized by ATG8e antibodies) at early stages (5 h), and a more significant colocalization at later stages of autophagy (10 h). Scale bars = 10 µm.

We also performed immunogold electron microscopy to confirm that double-membrane structures, characteristic of EXPO and autophagosomes, labeled positively for endogenous ATG8. Such structures were successfully labeled with GFP antibodies (raised in mice) in transgenic Arabidopsis plants or cells expressing either YFP-ATG8e or Exo70E2-GFP (Fig. 9, A–C). Through double immunogold labeling with ATG8e (raised in rats) and GFP antibodies (raised in mice), we also found examples for double-membrane structures in the cytosol apparently bearing both antigens (Fig. 9D). We also looked at the situation inside of the vacuole and, despite the partially degraded nature of the contents, were able to detect both GFP and ATG8e antigens on single membrane-bound structures (Fig. 9, E and F).

Figure 9.

Immunogold electron microscopy analysis of the distribution of Exo70E2 and ATG8 after autophagic induction in transgenic Arabidopsis. A and B, YFP-ATG8e recognized by GFP antibodies (raised in mice) was localized on the inner and outer membranes of autophagosomes in plants expressing YFP-ATG8e. C, Exo70E2-GFP labeled by GFP antibodies (raised in mice) localized to double-membrane structures similar to autophagosomes in cells expressing Exo70E2-GFP. D, In plants expressing Exo70E2-GFP, Exo70E2-GFP and ATG8 were recognized by GFP (raised in mice; arrowheads; 10 nm) and ATG8 antibodies (raised in rats; arrows; 6 nm) on the same double-membrane structure. E, Exo70E2-GFP recognized by GFP antibodies (raised in mice; arrowheads) was found on autophagic bodies in the vacuole lumen of cells expressing Exo70E2-GFP. F, In plants expressing Exo70E2-GFP, Exo70E2-GFP and endogenous ATG8 were recognized by GFP (raised in mice; arrowheads; 10 nm) and ATG8 antibodies (raised in rats; arrows; 6 nm) on the same autophagic bodies in the vacuole lumen. Scale bars = 500 nm.

Differential Membrane Recruitment of Exo70E2 and ATG8 during Autophagic Induction

Under normal conditions, ATG8 proteins mainly distribute into the cytosolic fraction due to its hydrophilic nature. However, the level of ATG8 proteins increases, and ATG8 proteins are recruited onto autophagosomes (membrane anchoring) upon autophagic induction (Ge et al., 2013; Zhuang et al., 2013). As Exo70E2 showed a gradual colocalization with ATG8 during autophagic induction (Figs. 3, 6, 7, and 8), we decided to determine whether Exo70E2 shows a similar membrane recruitment pattern as ATG8 upon autophagic induction. We therefore induced autophagy with BTH in transgenic Arabidopsis cells expressing either Exo70E2-GFP or YFP-ATG8f, and extracted proteins from both membrane and soluble fractions in the homogenates at different time points (0, 8, and 16 h after addition of BTH). As expected, YFP-ATG8f did indeed respond to autophagic induction: the membrane fraction of YFP-ATG8f greatly increased (indicated by the arrowhead in Fig. 10A, right blot). The soluble form of YFP-ATG8f also increased at both 8 and 16 h, suggesting new synthesis of YFP-ATG8f proteins and their transfer from the cytosol to the membrane of autophagosomes. However, the response of Exo70E2-GFP to autophagic induction is different (indicated by the arrow in Fig. 10A, left blot). There is no obvious signal increase in the blots of either the cytosol or membrane fractions, indicating that Exo70E2 does not respond to autophagy like ATG8f. Similar results were obtained from three repeated experiments. We then calculated the ratio of membrane fraction (CM) to total proteins (CS+CM) using results from these four independent experimental results, which confirmed the massive transfer of YFP-ATG8f from the cytosol to membrane at 8 h (Fig. 10B, right). Such a pattern could not be seen in Exo70E2-GFP samples (Fig. 10B, left). At 16 h, the ratio of membrane fraction (CM) to total proteins of YFP-ATG8f decreased (Fig. 10B, right). We did not inhibit the vacuolar degradation of autophagic bodies by adding ConcA, resulting in the relative decrease of membrane-bound YFP-ATG8f.

Figure 10.

Immunoblot analysis of protein dynamics of Exo70E2-GFP and YFP-ATG8f after autophagic induction. A, Immunoblot analysis of protein levels of Exo70E2-GFP (indicated by arrow) or YFP-ATG8f (indicated by arrowhead) in transgenic cells by GFP antibodies after BTH (100 µm) treatment at different time points. Anti-cFBPase and anti-ManI were used as loading controls for soluble protein and membrane protein fractions, respectively. B, Quantification of Exo70E2-GFP and YFP-ATG8f signals after autophagic induction. Values for the y coordinate were determined by cell membrane proteins (CM)/(cell-soluble proteins [CS]+CM). Different time points for sample collection were labeled on the x coordinate. The intensity of immunoblot bands was calculated using ImageJ software as previously described (Shen et al., 2013, 2014a).

DISCUSSION

Proteosomes, lysosomes, and autophagosomes are all involved in protein turnover in eukaryotic cells (Lecker et al., 2006; Dice, 2007; Eskelinen and Saftig, 2009). However, in response to stress conditions, in particular carbon or nitrogen deficiency, autophagosomal numbers and activity are considerably increased. Macroautophagy, which comprises both autophagosome formation and the subsequential degradation of cytosolic proteins and organelles in the lytic compartment of the cell, has been the subject of extensive research in yeast and mammalian systems (Yang and Klionsky, 2010; Feng et al., 2014), as well as in plants (Floyd et al., 2012; Liu and Bassham, 2012). Autophagosome formation is initiated by the appearance of a structure alternately termed the phagophore, phagophore assembly site, or isolation membrane (Rubinsztein et al., 2012). The origin of this cisternal-like structure in mammalian cells continues to be a very controversial issue, with claims of contributions from the endoplasmic reticulum (ER)-mitochondrial contact domains (Hamasaki et al., 2013), from the PM via clathrin-coated vesicles (Ravikumar et al., 2010), and from the ER-Golgi intermediate compartment (Ge et al., 2013; Ge and Schekman, 2014). However, it would seem that the true initiator of the phagophore seems to be a specialized domain of the ER (Hayashi-Nishino et al., 2009; Uemura et al., 2014), which is characteristically enriched in phosphatidylinositol 3-phosphate (Noda et al., 2010). A recent investigation on plants has also confirmed the ER as being the primary source of the phagophore membrane (Zhuang et al., 2013). Once the phagophore has been formed, it expands sealing its cargo in a double-membrane structure: the autophagosome.

A functional role for the exocyst complex in the autophagic process has recently been described. An Exo84-positive exocyst subcomplex has been shown to act as a scaffold bridging the upstream mechanistic target of rapamycin signaling cascade (Bar-Peled and Sabatini, 2012) and the downstream autophagic core machinery in a human epithelial cell line (Bodemann et al., 2011; Farré and Subramani, 2011). Similarly, in Arabidopsis, Exo70B1 has been reported to be essential for autophagosome formation and cargo transportation to the vacuole, indicating a possible conserved function of exocyst-mediated autophagic regulation (Kulich et al., 2013). On the other hand, AtExo70E2, a paralog of Exo70B1, is involved in unconventional protein secretion in plant cells and is recruited to a double-membrane structure that we have termed EXPO (Wang et al., 2010; Ding et al., 2012, 2014a, 2014b). The close morphological similarity between EXPO and autophagosomes raises the question as to whether these structures are more deeply related to one another, or might originate in the same way. Using YFP-ATG8e or YFP-ATG8f, the standard marker for autophagosomes, and specific antisera to ATG8e, we have shown here that, under normal (i.e. nonstarvation) conditions, there is no overlap in fluorescent signal with Exo70E2-GFP. However, we have made the unexpected discovery that, when autophagy is induced by different methods, EXPO no longer fuse with the PM but, like autophagosomes, are instead directed toward the vacuole. Inhibition of proteolytic degradation in the vacuole through ConcA during the autophagic induction treatment leads to the accumulation of autophagic bodies. The fluorescent signals for EXPO and autophagosomes in the autophagic bodies overlap more and more with the duration of autophagic induction. Therefore, we conclude that, although EXPO and autophagosomes are distinct organelles in normal cells, they appear to respond to autophagy in a similar manner.

There are several possible scenarios that can explain the vacuolar sequestration of structures bearing Exo70E2-GFP (see Fig. 11). (1) EXPO and autophagosomes fuse independently with the tonoplast and then fuse with one another in the lumen of the vacuole. For this to work, the PM SNARE fusion machinery of EXPO must be redirected for fusion with the tonoplast.

Figure 11.

Working model describing the relationship between EXPO and autophagosomes. Under normal conditions, EXPO sequestrate cytosolic cargo before delivering the cargo into the apoplast via unconventional protein secretion. However, upon autophagic induction, several scenarios are possible: (1) EXPO may fuse with autophagosomes after entry into the vacuole; (2) EXPO might fuse with autophagosomes before being internalized by the vacuole; or (3) Exo70E2 may be recruited to the phagophore membrane, which then develops into a hybrid autophagosome, before being sequestered in the vacuole.

(2) EXPO and autophagosomes fuse with one another in the cytosol, and the hybrid structures then fuse with the tonoplast. Again, alterations in the SNARE fusion machinery of EXPO and/or autophagosomes would be required, since under nonstarvation conditions these organelles remain separate entities.

(3) With the onset of autophagy, Exo70E2 is recruited together with ATG8 to the phagophore membrane. The resulting hybrid autophagosome then fuses with the tonoplast. Thus, an explanation must be found for the nonfunctional recruitment of Exo70E2 to autophagosomes during starvation, especially since Exo70E2 expression is not elevated during autophagic induction.

The unusual images obtained with FM4-64 staining deserve comment. First, the visualization of punctate fluorescent structures in the lumen of the vacuole is not specifically related to the use of FM4-64, since identical results were obtained with standard fluorescent tonoplast markers (Supplemental Fig. S6). Second, even without inducing autophagy through BTH treatment, ConcA caused FM4-64 and Exo70E2-positive punctae to appear in the vacuole of transgenic Arabidopsis root cells. How do these fluorescent punctae relate to autophagy? ConcA is an antibiotic that is known to target the Golgi and trans-Golgi network (Robinson et al., 2004; Dettmer et al., 2006; Scheuring et al., 2011). Thus, the appearance of FM4-64 punctae in the lumen of the vacuole has nothing to do with conventional secretory membrane trafficking to the vacuole, which is blocked by ConcA. In any case, fusion of single-membrane-bound transport vesicles with the tonoplast would not lead to the formation of discrete single-membrane-bound autophagic bodies. FM4-64-positive structures in the vacuole can therefore only arise through fusion of double-membrane organelles (e.g. autophagosomes or EXPO) with the tonoplast or through invagination of the tonoplast and subsequent fission (Maîtrejean and Vitale, 2011). Although we have demonstrated the vacuolar internalization of fluorescent EXPO and autophagosomal markers, both of which are double-membrane structures, FM4-64-positive punctae in the vacuole appear earlier than EXPO signals. Moreover, the number of FM4-64 punctae appear to exceed those labeled by EXPO or autophagosomal markers. Therefore, we tentatively conclude that some of the vacuolar FM4-64 punctae do represent a form of tonoplast turnover, which is only made visible through ConcA preventing acidification and proteolysis in the vacuole.

MATERIALS AND METHODS

Plant Materials and Growth Condition

Transgenic cells used in this study were generated and maintained as in previous reports (Wang et al., 2010; Zhuang et al., 2013). Arabidopsis (Arabidopsis thaliana) transgenic plants expressing either YFP-ATG8e or YFP-RABG3f were generated as previously described (Zhuang et al., 2013; Cui et al., 2014). Arabidopsis transgenic plants expressing YFP-VAMP711 were obtained from the European Arabidopsis Stock Center (WAVE_9Y; Geldner et al., 2009). For Exo70E2-XFP transgenic plants, full-length coding sequence of Exo70E2 was amplified and cloned into pBI121 backbone for construction of the XFP fusion (Wang et al., 2010; Ding et al., 2014b). Exo70E2-RFP transgenic plants were then crossed into YFP-ATG8e transgenic plants, and the T1 generation with double fluorescent signals was screened out for further study. Procedures for generating and screening Arabidopsis transgenic plants were performed as described previously (Zhang et al., 2006). Transgenic seeds were surface sterilized and sown on plates with Murashige and Skoog salts plus 1% (w/v) Suc and 0.8% (w/v) agar. The seeded plates were kept at 4°C for 3 d before being moved to the growth chamber at 22°C under a long-day (16-h light/8-h dark) photoperiod.

Autophagic Induction in Transgenic Cells and Plants

For Suc starvation, 3-d-old Arabidopsis transgenic cells were collected by natural precipitation, and supernatant was discarded. The cell pellets were washed with an equal volume of Suc-free culture medium twice. Cells were then incubated in Suc-free medium at a rotation of 110 rpm. For BTH and ConcA treatments, transgenic cells or plants were incubated in culture medium with vector methanol (1:200) for mock control, or in medium containing 100 µm BTH plus 0.5 µm ConcA for 8 h (early stage for cells) or 5 h (early stage for plants), or 16 h (late stage for cells) or 10 h (late stage for plants) before observation and fixation. Nitrogen starvation was performed by transferring the 5-d-old seedlings to nitrogen-free Murashige and Skoog medium plus 0.5 µm ConcA for 12 h (early stage) or 24 h (late stage).

Transient Expression

Transient expression was performed as described previously (Miao and Jiang, 2007; Shen et al., 2014b). After electroporation, transfected protoplasts were incubated at 26°C in protoplast culture medium (Shen et al., 2014b) before confocal observation at different time points. Confocal fluorescence images were captured using the Leica SP8 laser scanning confocal system. Detailed confocal setting was described elsewhere (Gao et al., 2012, 2014; Zhao et al., 2015). For each expressing construct, more than 20 individual cells were examined that represented >75% of the cells showing the same patterns or localizations. Pearson correlation coefficients were calculated using a Pearson and Spearman Correlation coefficients colocalization plug-in of the ImageJ program (National Institutes of Health; French et al., 2008; Wang et al., 2014; Gao et al., 2015). Images were processed via Adobe Photoshop as described previously (Jiang and Rogers, 1998).

Immunofluorescent Staining

Three-day-old Arabidopsis transgenic cells expressing either Exo70E2-GFP or YFP-ATG8f, or 5-d-old Arabidopsis transgenic plants expressing either Exo70E2-GFP or YFP-ATG8e were subjected to autophagic induction, respectively. Different samples were collected at different time points of treatment and then fixed as previously described (Sauer et al., 2006; Wang et al., 2010). Polyclonal antibodies against Exo70E2 raised in rabbits and ATG8e raised in rats were generated as described previously (Wang et al., 2010; Zhuang et al., 2013) and used at a concentration of about 4 μg mL⁻¹. For secondary antibodies, Alexa Fluor-568 anti-rabbit or anti-rat (Molecular Probes) was used at 1:1,000 dilutions for immunodetection. Pearson correlation coefficients were calculated from more than five individual cells by using a PSC colocalization plug-in of the ImageJ program (French et al., 2008; Wang et al., 2014; Gao et al., 2015), which produced r values in the range of −1 to +1, with 0 indicating no discernible correlation, and +1 and −1 indicating strong positive or negative correlations, respectively.

FM4-64 Uptake Study

FM4-64 uptake experiments were performed basically as previously described (Tse et al., 2004; Lam et al., 2007, 2008). Transgenic Arabidopsis plants expressing either Exo70E2-GFP or YFP-ATG8e were treated by autophagic induction, described above, and labeled with FM4-64 (12 µm) for 3 h before confocal imaging to highlight the tonoplast staining (Cai et al., 2014). Images were collected at different time points as indicated.

Three-Dimensional Confocal Imaging and Related Projection Reconstruction by Imaris

Three-dimensional confocal imaging was performed as described previously (Wang et al., 2010) with some modification. Confocal imaging of multiple optical sections was conducted at the z axis with 0.4 μm per step and then used for three-dimensional movie construction using Imaris software following instructions provided by the manufacturer (Bitplane).

Protein Extraction and Immunoblot Analysis

After BTH treatment, protein extractions from transgenic cells were performed as described previously, with minor modification (Shen et al., 2013, 2014a). Cells were broken down using a cell disruptor, and whole-cell lysates were extracted using the 5X extraction buffer (250 mm Tris-HCl [pH 7.4], 750 mm NaCl, 5 mm EDTA, 1X Complete Protease Inhibitor Cocktail from Roche). For separation of CS and CM fractions, low-speed centrifugation was first applied at 600g for 3 min at 4°C to precipitate cell debris. Supernatants were transferred to a new tube followed by high-speed centrifugation at 100,000g for 30 min at 4°C. Then, supernatants were collected as CS fraction, and pellets containing CM proteins were resuspended with 1X extraction buffer. Proteins were separated on 10% polyacrylamide gels by SDS-PAGE and immunodetected with GFP, cFBPase (Agrisera, AS04 043), and ManI (Tse et al., 2004) antibodies. Statistical analysis was performed by analyzing four independent experimental results. Anti-cFPBase signals (indicator of soluble fractions) or anti-ManI signals (indicator of membrane fractions) at both 8 and 16 h were divided by those signals at 0 h (considered as 100%), respectively, to obtain corresponding normalization factors. Then, signals from either Exo70E2-GFP or YFP-ATG8f fusion proteins were divided by corresponding normalization factors, and the ratio of membrane fraction (CM) to total proteins (CS+CM) was calculated and compared with the value at 0 h to show the relative change.

High Pressure Freezing and Immunogold Labeling

Transgenic cells or plants were first treated to induce autophagy. High pressure freezing and subsequent freezing substitution were performed as previously described (Jia et al., 2013; Wang et al., 2013). Transmission electron microscopy examination was performed via a Hitachi H-7650 transmission electron microscopy with a MacroFire monochrome CCD camera (Optronics) operating at 80 kV. Immunogold labeling for ultrathin sections was performed using ATG8e antibodies raised in rats at 80 μg mL⁻¹, and GFP antibodies raised in mice at 60 μg mL⁻¹ before gold-coupled secondary antibodies at 1:50 dilution.

The Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are as follows: Exo70E2 (AT5G61010), ATG8e (AT2G45170), ATG8f (AT4G16520), RABG3f (AT3G18820), and VAMP711 (AT4G32150).

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Specificity of Exo70E2 and ATG8e antibodies used in this study.

• Supplemental Figure S2. Statistical analysis of colocalization efficiency between EXPO and autophagosomes under different conditions.

• Supplemental Figure S3. Turnover of EXPO and autophagosomes in response to both Suc and nitrogen starvation.

• Supplemental Figure S4. Quantification of Exo70E2-GFP or YFP-ATG8e/f signals inside the vacuole under different conditions.

• Supplemental Figure S5. Statistical study of fluorescent signal distribution of Exo70E2, ATG8e/f, or FM4-64 under different conditions.

• Supplemental Figure S6. Different tonoplast markers show partial colocalization with FM4-64 inside the vacuole due to autophagy.

• Supplemental Figure S7. Identity of vacuolar lumen located fluorescent punctae caused by autophagic induction is further proven by three-dimensional confocal imaging.

• Supplemental Figure S8. Exo70E2-mRFP gradually colocalizes with YFP-ATG8e.

• Supplemental Movie S1. Three-dimensional image reconstruction (by Imaris software) of an Arabidopsis transgenic plant expressing Exo70E2-GFP subjected to autophagic induction and FM4-64 uptake.

• Supplemental Movie S2. Three-dimensional image reconstruction (by Imaris software) of an Arabidopsis transgenic plant expressing YFP-ATG8e subjected to autophagic induction and FM4-64 uptake.

• Supplemental Movie S3. Three-dimensional image reconstruction (by Imaris software) of an Arabidopsis transgenic plant expressing YFP-RABG3f subjected to autophagic induction.

• Supplemental Movie S4. Three-dimensional image reconstruction (by Imaris software) of an Arabidopsis transgenic plant expressing YFP-VAMP711 subjected to autophagic induction.

Glossary

PM

plasma membrane

EXPO

exocyst-positive organelles

ConcA

concanamycin A

CaMV

Cauliflower mosaic virus

BTH

benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester

CS

cell-soluble proteins

CM

cell membrane proteins

ER

endoplasmic reticulum