A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis

Abstract

The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1(d)[NI]) of Arabidopsis thaliana RAB-E1(d) and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1(d)[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1(d)[NI] caused less intracellular secGFP accumulation than RAB-D2(a)[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2(a)[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1(d)[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)-tagged Arabidopsis RAB-F2(b). Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1(d)[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1(d), YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1(d)[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.

Figure 1.

A Dominant-Inhibitory Mutant in RAB-E1^d Results in Increased Intracellular secGFP Accumulation. (A) Time course of GFP-HDEL and secGFP fluorescence during transient expression in tobacco leaf epidermal cells infected with the respective Agrobacterium strains. (B) to (F) Confocal images at low magnification of tobacco leaf epidermal cells expressing secGFP (B), GFP-HDEL (C), secGFP+RAB-D2^a[NI] (D), secGFP+RAB-E1^d (E), and secGFP+RAB-E1^d[NI] (F). Bar in (F) = 100 μm for (B) to (F). (G) Relative GFP fluorescence intensity of tobacco leaf epidermal cells expressing GFP-HDEL, secGFP, secGFP + RAB-E1^d, secGFP + RAB-E1^d[NI], and secGFP + RAB-D2^a[NI].

Figure 2.

secGFP Labels ER and Post-ER Punctate Structures in the Presence of RAB-E1^d[NI]. (A) to (D) Confocal images at high magnification of tobacco leaf epidermal cells expressing secGFP (A), GFP-HDEL (B), secGFP + RAB-D2^a[NI] (C), and secGFP + RAB-E1^d[NI] (D). Note the punctate structures indicated by arrowheads in the image (D) and the inset. Bar in (D) = 10 μm for (A) to (D). (E) Average number of fluorescent cells and frequency of particular intracellular fluorescence patterns per field of view in cells expressing secGFP, secGFP + RAB-E1^d[NI], and secGFP + RAB-D2^a[NI]. n is the number of cells scored.

Figure 3.

Expression of RAB-E1^d[NI] Does Not Directly Affect ER-to-Golgi Trafficking. (A) to (C) Confocal images of a tobacco epidermal cell expressing secGFP (A) and YFP-HDEL (B) in presence of RAB-E1^d[NI]. (C) was obtained by merging images (A) and (B). Note that the punctate compartments labeled by secGFP fluorescence (arrows) are not marked by YFP-HDEL fluorescence. Note also the existence of a second type of punctate structure that is faintly labeled by secGFP but excludes YFP-HDEL (arrowheads in [C]). Bar in (C) = 10 μm for (A) to (C). (D) Enlarged image of the confocal image in (C) showing details of small but bright (arrows) and apparently larger but faint (arrowheads) punctate structures labeled by secGFP but not by YFP-HDEL in the presence of RAB-E1^d[NI]. (E) and (F) Merged GFP/YFP confocal images of tobacco leaf epidermal cells expressing GFP-HDEL + YFP-HDEL in the presence of RAB-E1^d[NI] (E) and secGFP + YFP-HDEL in the presence of RAB-D2^a[NI] (F). Bar in (F) = 5 μm for (D) to (F). (G) to (I) Confocal images of tobacco leaf epidermal cells expressing ST-YFP (G), ST-YFP + RAB-E1^d[NI] (H), and ST-YFP + RAB-D2^a[NI] (I). Bar in (I) = 10 μm for (G) to (I).

Figure 4.

In the Presence of RAB-E1^d[NI], secGFP Accumulates in the Golgi Stacks and in a PVC. (A) to (F) Confocal images of a tobacco epidermal cell expressing secGFP ([A] and [D]) and ST-YFP ([B] and [E]) in the presence of RAB-E1^d[NI]. (C) is a merge of (A) and (B). (D) to (F) are enlarged images of (A) to (C), respectively. Note the colocalization of ST-YFP fluorescence with the large, faint secGFP punctate structures (arrowheads in [D] to [F]). The small, bright secGFP punctate structures (arrows image [D] and [F]) do not colocalize with ST-YFP. Bar in (C) = 10 μm for (A) to (C). (G) to (I) Confocal images of a control tobacco epidermal cell coexpressing ST-YFP and RAB-E1^d[NI] without secGFP. Imaging parameters were identical to those used for (A) to (F). No signal is visible in the GFP image (G) excluding the possibility that the Golgi-sized faint punctate structures represent bleed-through of YFP fluorescence into the GFP detection channel in (A) and (D). Bar in (I) = 5 μm for (D) to (I). (J) to (L) Confocal images of an FM4-64–stained tobacco leaf epidermal cell from a region coinfiltrated with secGFP and RAB-E1^d[NI]. (L) is a merged image from the secGFP image (J) and the FM4-64 image (K). White arrows identify examples of small brightly labeled secGFP punctate structures marked by FM4-64 (K). Red arrow indicates a structure apparently labeled by FM4-64 alone. Bar in (L) = 2 μm for (J) to (L). (M) to (O) Confocal images of a tobacco leaf epidermal cell coexpressing secGFP and YFP-RAB-F2^b in the presence of RAB-E1^d[NI]. (O) was obtained by merging the secGFP image (M) and YFP-RAB-F2^b image (N). Arrows indicate examples of small brightly labeled secGFP punctate structures that are labeled by YFP-RAB-F2^b fluorescence (N). Bar in (O) = 1 μm for (M) to (O).

Figure 5.

RAB-E1^d[NI] Does Not Alter the Distribution of the Vacuole-Targeted Marker spo:GFP. Confocal images of tobacco epidermal cells expressing spo:GFP (A), spo:GFP + RAB-E1^d[NI] (B), and spo:GFP + RAB-D2^a[NI] (C). Bar in (C) = 10 μm for (A) to (C).

Figure 6.

secGFP and aleu-GFP Are Transported to the Vacuole in the Presence and Absence of RAB-E1^d[NI]. Agrobacterium harboring pVKH-secGFP was infiltrated into tobacco leaves at OD₆₀₀ 0.03 (three times the usual titre), and the aleu-GFP strain was infiltrated at OD₆₀₀ 0.1 as described previously (Kotzer et al., 2004). All samples were treated with E64-d in darkness for 12 to 14 h and were imaged ∼50 h after inoculation with Agrobacterium. Asterisks identify cells that express little or no GFP and indicate the background vacuolar fluorescence in these images. Bars = 50 μm. (A) to (D) Medial confocal sections through abaxial epidermal cells indicating secGFP fluorescence in E64-d–treated leaves. (A) secGFP only; (B) secGFP coinfiltrated with RAB-E1^d; (C) secGFP coinfiltrated with RAB-E1^d[NI]; (D) secGFP coinfiltrated with RAB-D2^a[NI]. (E) to (N) Location of aleu-GFP in the presence of dominant-inhibitory Rab GTPase mutants in single optical sections ([E], [G], [I], [K], and [M]) through abaxial epidermal cells and in projections ([F], [H], [J], [L], and [N]) of series of optical sections on the z axis through the outer cortical region of abaxial epidermal cells. (E) and (F) aleu-GFP alone; (G) and (H) aleu-GFP + RAB-E1^d; (I) and (J) aleu-GFP + RAB-E1^d[NI]; (K) and (L) aleu-GFP + RAB-F2^b[SN]; (M) and (N) aleu-GFP + RAB-D2^a[NI]. Arrows indicate regions of apoplast between infected cells that exhibit clear aleu-GFP fluorescence in (E) to (H) but are weakly fluorescent in (I) and (J).

Figure 7.

RAB-E1^d Targets YFP to the Golgi via a GTP- and Prenylation-Dependent Mechanism. (A) to (C) Confocal analysis of cells coexpressing YFP-RAB-E1^d (A) and ST-GFP (B). (C) is merged from the YFP-RAB-E1^d image (A) and the ST-GFP image (B). Note the precise colocalization of YFP-RAB-E1^d–labeled punctate structures and Golgi (insets). (D) to (F) Confocal analysis of tobacco epidermal cells coexpressing YFP-RAB-E1^d[SN] (D) and ST-GFP (E). (F) was obtained by merging images (D) and (E). Note the exclusively cytosolic YFP-RAB-E1^d[SN] fluorescence in (D). (A) to (I) are projections on the z axis of a series of optical sections of the cortical cytoplasm. Bar in (I) = 25 μm for (A) to (I). (G) to (I) Confocal analysis of tobacco epidermal cells coexpressing YFP-RAB-E1^dΔCC (G) and ST-GFP (H). (I) was obtained by merging (G) and (H). Like YFP-RAB-E1^d[SN], YFP-RAB-E1^dΔCC fluorescence is exclusively cytosolic (G). Insets in (D) to (I) show regions of each image at higher resolution, confirming that YFP-tagged RAB-E1^d mutants do not colocalize with Golgi stacks. (J) to (L) Confocal analysis of a tobacco epidermal cell coexpressing YFP-RAB-D2^a (J) and ST-GFP (K). (L) was obtained by merging (J) and (K). Arrows identify examples of YFP-RAB-D2^a–labeled punctate structures that are distinct from the Golgi stacks. (M) to (O) Confocal analysis of a tobacco epidermal cell coexpressing YFP-RAB-D2^a[SN] (M) and ST-GFP (N). (O) was obtained by merging (M) and (N). Note the exclusive cytosolic YFP-RAB-D2^a[SN] fluorescence in (M). (P) to (R) Confocal images of a control tobacco epidermal cell expressing ST-GFP. The images were captured with identical confocal laser scanning parameters used for (J) to (O). (R) is merged from (P) and the ST-GFP image (Q). The absence of signal in the YFP image (P) excludes the possibility that the Golgi-sized faint punctate structures in (J) represented bleed-through of GFP fluorescence into the YFP detection channel. Bar in (R) = 5 μm for (J) to (R).

Figure 8.

YFP-Tagged RAB-E1^d Does Not Label the PVC That Accumulates secGFP. (A) to (C) Confocal images of a tobacco epidermal cell coexpressing secGFP and YFP-RAB-E1^d at ∼50 h after infiltration, when secGFP accumulation is highest (Figure 1A). (C) is the merged image of secGFP fluorescence (A) and YFP-RAB-E1^d fluorescence (B). White arrows in (A) to (C) and insets indicate examples of small, bright punctate structures that are labeled by secGFP but not by YFP-RAB-E1^d. Red arrows indicate examples of large, faint Golgi punctate structures that are labeled by YFP-RAB-E1^d fluorescence. (D) to (F) Confocal images of a control tobacco epidermal cell expressing YFP-RAB-E1^d without secGFP. The images were captured with identical image settings used for (A) to (C). (F) is merged from (D) and the YFP-RAB-E1^d image (E). The absence of signal in the GFP image (D) excludes the possibility that the Golgi-sized faint punctate structures represent bleed-through of YFP fluorescence into the GFP detection channel. Bar in (F) = 5 μm for (A) to (F); bar in inset (C) = 2 μm for all insets.

Figure 9.

The Default Pathway to the Apoplast Does Not Involve the RAB-F2^b-Positive PVC. Confocal analysis of tobacco epidermal cells coexpressing secGFP (A) and secRFP (B). (C) was obtained by merging (A) and (B). Both secGFP (A) and secRFP (B) can be visualized in the ER, but the PVC labeled by secGFP (arrows in [A], [C], and insets) is exclusive of secRFP (arrows in [C] and insets). secRFP was clearly observed in the apoplast where it was stable (B). Bar in (C) = 10 μm for (A) to (C); bar in inset (C) = 3 μm for all insets.