MAIGO5 functions in protein export from Golgi-associated endoplasmic reticulum exit sites in Arabidopsis

Abstract

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A-green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.

Figure 1.

Isolation of an Arabidopsis Mutant, mag5, That Exhibits Abnormal Accumulation of Precursors of Two Major Storage Proteins in Seeds. (A) Immunoblot of dry seeds with anti-12S globulin and anti-2S albumin antibodies. Wild-type (WT) seeds accumulated mature forms of the major storage proteins 12S globulin (12S) and 2S albumin (2S). mag5 mutant (mag5-1) seeds accumulated precursors (p12S and p2S) of storage proteins, as did other known mag mutants (mag2-1 and mag4-1). (B) Protein profiles of dry seeds of wild-type, mag5-1, mag2-1, and mag4-1 plants on an SDS gel stained with Coomassie blue. Molecular masses of the precursor forms (p12S and p2S) are indicated on the right in kilodaltons, and those of the markers are indicated on the left. 12S-α and 12S-β, 12S globulin subunits; 2S-L and 2S-S, 2S albumin subunits.

Figure 2.

MAG5 Encodes a Putative Ortholog with Low Sequence Similarity to Human Sec16A and Yeast Sec16p. (A) Schematic representation of the MAG5/SEC16A gene (At5g47480) and the homologous SEC16B gene (At5g47490). Three mag5 mutants (mag5-1, mag5-2, and mag5-3) and three sec16b mutants (sec16b-1, sec16b-2, and sec16b-3) are shown, and each mutation site is indicated. F1-3 and R1-3 indicate the primers used in (B) and (C). Closed boxes indicate protein-coding regions, open boxes indicate untranslated regions, and solid lines indicate introns. WT, the wild type. (B) RT-PCR using the F1 and R1 primers shows that a 34-bp deletion in the MAG5 gene leads to a smaller transcript in the mag5-1 mutant than in the wild type. (C) RT-PCR of the MAG5/SEC16A and SEC16B transcripts in rosette leaves of the wild-type and mutant plants. The F2 and R2 primers were used to amplify MAG5/SEC16A, whereas the F3 and R3 primers were used to amplify SEC16B. ACT2 was included as a control. (D) Immunoblot of dry seeds with anti-12S globulin and anti-2S albumin antibodies. Each of the mag5 mutants accumulated the storage protein precursors (p12S and p2S), whereas the sec16b mutants did not.12S-α, a 12S globulin subunit; 2S-S and 2S-L, 2S albumin subunits.

Figure 3.

mag5 Seed Cells Develop Abnormal Structures with Electron-Dense Cores Containing 2S Albumin and a Matrix Containing 12S Globulin. (A) Electron micrographs of seed cells of wild-type (WT) and mag5 mutant (mag5-1, mag5-2, and mag5-3) plants. All mag5 mutants developed abnormal structures, designated MAG bodies, as indicated by arrowheads. Bars = 2 µm. (B) Immunoelectron micrographs of seed cells of wild-type and mag5 mutant plants using the anti-12S globulin and anti-2S albumin antibodies. Bars = 0.5 µm.

Figure 4.

Lack of MAG5/SEC16A Causes an Abnormal ER Conformation and Interferes with Protein Export. (A) Confocal images of wild-type (WT) epidermal cells of cotyledons expressing the Golgi marker ST-GFP (left panel). mag5-2 epidermal cells of cotyledons expressing the Golgi marker ST-GFP, which is partially retained in the ER (right panel). The arrow and arrowhead indicate Golgi and ER, respectively. Bar = 5 μm. (B) and (C) FRAP analyses of ST-GFP in Golgi stacks at the cortex of cotyledon epidermal cells in wild-type and mag5-2/ST-GFP seedlings. The fluorescence recovery half-time was significantly slower in mag5-2 seedlings than in wild-type seedlings (P < 0.05) (B). The mobile fraction (%) did not significantly differ between wild-type and mag5-2 seedlings (P > 0.05) (C). Sample size in each experiment (i.e., number of bleached Golgi stacks) = 10. Error bars indicate sd.

Figure 5.

Punctate Structures Labeled by MAG5/SEC16A Are Surrounded by the Cup-Shaped Regions of the ER That Face Associated Golgi Stacks. (A) Immunoblot of individual T2 seed grains from seven independent T1 lines (ProMAG5/SEC16A:MAG5/SEC16A-GFP in mag5-1) using anti-12S globulin and anti-2S albumin antibodies. The mag5-1 phenotype of abnormal accumulation of precursors was rescued in these lines. WT, the wild type. (B) Confocal image of epidermal cells of cotyledons in T2 line number two shown in (A). MAG5/SEC16A-GFP is distributed in the cytosol and puncta (arrows). Bar = 5 µm. (C) Confocal images of transiently transformed tobacco epidermal cells coexpressing GFP-MAG5/SEC16A and the cis-Golgi marker ERD2-YFP. The degree of colocalization between the two markers was analyzed using Zeiss software in the spot highlighted by the white circle and enlarged in the inset. The graph shows that the fluorescence signals partially overlap; however, the fluorescence peaks are shifted. In the graph, distance is presented in pixels (1 pixel = 0.09 µm) (D) Colocalization of GFP-MAG5/SEC16A and the trans-Golgi marker ST-mRFP was analyzed using Zeiss software in the spot highlighted by the white circle and enlarged in the inset. The graph shows that the fluorescence signals do not overlap and the fluorescence peaks are even more shifted than in (C). In the graph, distance is presented in pixels (1 pixel = 0.09 µm) (E) Confocal images of transiently transformed tobacco epidermal cells coexpressing GFP-MAG5/SEC16A and ER-YK. The degree of colocalization between the two markers was analyzed using Zeiss software in the spot highlighted by the white circle and enlarged in the inset (arrow). The graph shows a high degree of colocalization between these two markers, and their fluorescence peaks coincide (arrow). In the graph, distance is presented in pixels (1 pixel = 0.09 µm). Bars = 5 µm (main image) and 1 µm (inset). (F) Three-dimensional projection reconstruction and rendering of images of cells expressing the same markers as shown in (E). In this projection, the ER is cup shaped around MAG5/SEC16A-positive structures, supporting the hypothesis that MAG5/SEC16A is associated with specific regions of ER. Bar = 1 µm

Figure 6.

MAG5/SEC16A Interacts with SEC13 and SEC31. (A) and (B) Immunoblots (A) and silver staining (B) of anti-GFP antibody immunoprecipitate samples from transgenic plants expressing GFP or MAG5/SEC16A-GFP. The arrowhead indicates full-length MAG5/SEC16A-GFP. (C) Identification of MAG5/SEC16A-interacting proteins by mass spectrometry. The Arabidopsis Genome Initiative (AGI) codes and annotations were obtained from The Arabidopsis Information Resource database (http://www.Arabidopsis.org). Scores were calculated by Mascot (Matrix Science). (D) Yeast two-hybrid analysis of the AH109 strain expressing a fusion protein containing the GAL4 DNA binding domain (BD) and a fusion protein containing the GAL4 activation domain (AD). Tenfold dilution series of transformants were incubated on SD/–Leu/–Trp/–His/–Ade medium (–LWHA) or SD/–Leu/–Trp medium (–LW). Negative controls (empty vector) were also included.

Figure 7.

MAG5/SEC16A Fusion Proteins Colocalize with YFP-SEC24A and SEC13A-GFP. (A) Confocal images of tobacco leaf epidermal cells coexpressing GFP-MAG5/SEC16A and YFP-SEC24A. GFP-MAG5/SEC16A is distributed in the cytosol and at puncta that colocalize with YFP-SEC24 (arrows). The inset corresponds to an enlarged region of the main image. Open arrows indicate chloroplasts, which are visible because of chlorophyll autofluorescence in the GFP channel. Bars = 5 µm (main image) and 2 µm (insets). (B) Confocal images of tobacco leaf epidermal cells coexpressing MAG5/SEC16A-tagRFP and SEC13A-GFP. MAG5/SEC16A-tagRFP colocalizes with SEC13A-GFP. Bars = 5 µm (main image).

Figure 8.

Comparison of the Dynamics of MAG5/SEC16A with That of SEC24 at ERESs. (A) and (B) Confocal images of leaf epidermal cells of mag5-1 rescued by expression of ProMAG5/SEC16A:MAG5/SEC16A-GFP (A) and of leaf epidermal cells of Arabidopsis plants expressing YFP-SEC24A (B). ROI, region of interest. Bar = 5 µm. (C) and (D) Example regions of interest in (A) and (B), respectively, were used to obtain curves of FRAP events. (E) and (F) Distributions of the individual half-time measurements (E) and mobile fractions (F) are shown. The data represent the mean of 13 to 15 independent bleaching experiments (horizontal bars). Samples were treated with latrunculin B to stop movement of ERESs, as described previously (Brandizzi et al., 2002b). (G) and (H) Confocal images of tobacco leaf epidermal cells expressing GFP-MAG5/SEC16A (G) or YFP-SEC24 (H). Bars = 5 µm. (I) and (J) Example ROI in (G) and (H), respectively, were used to obtain curves of FRAP events. (K) and (L) Distributions of the individual half-time measurements (K) and mobile fractions (L) are shown. The data represent the mean of 13 to 17 independent bleaching experiments (horizontal bars). Samples were treated as described in (E) and (F).

Figure 9.

A Deficiency of MAG5/SEC16A Does Not Affect the Association of COPII Components with Membranes. (A) Immunoblot of subcellular fractions generated from 5-d-old wild-type (WT) and mag5-1 seedlings showing the distribution of SEC13A-GFP. T, total extract, P1, pellet obtained following centrifugation at 1000g ; P100, pellet obtained following centrifugation at 100,000g; S100, supernatant obtained following centrifugation at 100,000g. Antibodies against GFP, VSR1 (membrane fraction marker), and ALEU (soluble fraction marker) were used. (B) Quantitative analysis of the ratio of the SEC13A-GFP signal intensity in P1 plus P100 compared with that in S100 in the immunoblot shown in (A). The data represent the mean of three independent experiments (Student’s t test, P > 0.05, n = 3). (C) Immunoblot of each fraction showing the distribution of YFP-SEC24A in wild-type and mag5-1 seedlings. (D) Quantitative analysis of the ratio of YFP-SEC24A signal intensity in P1 plus P100 compared with that in S100 in the immunoblot shown in (C). The data represent the mean of three independent experiments (Student’s t test, P > 0.05, n = 3). (E) Immunoblot of each fraction showing the distribution of SAR1 in wild-type and mag5-1 seedlings. An anti-SAR1 antibody was used. (F) Quantitative analysis of the ratio of the SAR1 signal intensity in P1 plus P100 compared with that in S100 in the immunoblot shown in (E). The data represent the mean of three independent experiments (Student’s t test, P > 0.05, n = 3).

Figure 10.

MAG5/SEC16A Functions in the Dynamic Cycling of SEC13 and SEC24 on and off ERESs (A) Confocal images of SEC13A-GFP in leaf epidermal cells of wild-type (WT) and mag5-1 plants. The bleached area is shown in the circle. Bar = 5 µm. (B) Confocal images of YFP-SEC24A in leaf epidermal cells of wild-type and mag5-1 plants. The bleached area is shown in the circle. Bar = 5 µm. (C) Distribution of the individual half-times of SEC13A-GFP in wild-type and mag5-1 plants. The data represent the mean of 27 to 29 independent bleaching experiments following treatment with latrunculin B to stop movement of ERESs. The mean half-time of SEC13A-GFP is 3.61 ± 0.19 s and 2.03 ± 0.13 s in wild-type and mag5-1 plants, respectively (P < 0.0001). (D) Distribution of the individual half-time measurement for YFP-SEC24A in wild-type and mag5-1 plants. The data represent the mean of 33 to 37 independent bleaching experiments. The mean half-time of YFP-SEC24A is 3.23 ± 0.08 s and 2.84 ± 0.07 s in wild-type and mag5-1 plants, respectively (P < 0.005). (E) Analysis of the mobile fraction of SEC13A-GFP in wild-type and mag5-1 plants, showing that the amount of SEC13-GFP recovered after photobleaching does not significantly differ between wild-type and mag5-1 plants. The mean mobile fraction of SEC13A-GFP is 81% ± 1.6% and 77.19% ± 2.4% in wild-type and mag5-1 plants, respectively (P > 0.05). (F) Analysis of the mobile fraction of YFP-SEC24A in wild-type and mag5-1 plants, showing the amount of YFP-SEC24A recovered after photobleaching does not significantly differ between wild-type and mag5-1 plants. The mean mobile fraction of YFP-SEC24A is 70.70% ± 2.3% and 64.79% ± 2.05% in wild-type and mag5-1 plants, respectively (P > 0.05).