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Unique COPII Component Atsar1a/Atsec23a Pair Is Required for the Distinct Function of Protein ER Export in Arabidopsis Thaliana

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Unique COPII Component Atsar1a/Atsec23a Pair Is Required for the Distinct Function of Protein ER Export in Arabidopsis Thaliana Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana Yonglun Zenga, Kin Pan Chunga, Baiying Lia, Ching Man Laia,b, Sheung Kwan Lamc, Xiangfeng Wanga, Yong Cuia, Caiji Gaoa, Ming Luoa, Kam-Bo Wongb, Randy Schekmanc, and Liwen Jianga,d,1 aSchool of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China; bSchool of Life Sciences, Center for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China; cDepartment of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and dShenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518057, China Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved October 9, 2015 (received for review October 3, 2015) Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi proteins, except for Sec13. Elucidating the reason for the existence via the coat protein complex II (COPII) vesicle, which consists of five and conservation of multiple paralogs in higher organisms poses a cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, major challenge. Recently, mutations of some COPII paralogs COPII transport has diversified due to gene duplication, creating including Sec23A, Sec23B, and Sec24B have been shown to cause multiple COPII paralogs. Evidence has accumulated, revealing the distinct developmental disorders and human diseases (8). More functional heterogeneity of COPII paralogs in protein ER export. interestingly, even though sharing high sequence identity with Sar1B, the small GTPase of COPII machinery, seems to be specialized Sar1A, human Sar1B seems to be specialized to enable the ER for large cargo secretion in mammals. Arabidopsis contains five Sar1 export of large cargo chylomicrons (9). Different mutations in and seven Sec23 homologs, and AtSar1a was previously shown to Sar1B are associated with lipid absorption disorders due to defects exhibit different effects on α-amylase secretion. However, mecha- in chylomicron secretion: Anderson disease and chylomicron re- nisms underlying the functional diversity of Sar1 paralogs remain tention disease (9). However, mechanism underlying the distinct unclear in higher organisms. Here, we show that the Arabidopsis functions of human Sar1 homologs remains unclear. PLANT BIOLOGY Sar1 homolog AtSar1a exhibits distinct localization in plant cells. In Arabidopsis, COPII isoforms are greater in number than in Arabidopsis Transgenic plants expressing dominant-negative other eukaryotes. There are five Sar1, two Sec13, two Sec31, seven AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis Sec23, and three Sec24 isoforms (10). However, the significance of analysis identified a single amino acid, Cys84, as being responsible this diversification remains unresolved, raising the question as to for the functional diversity of AtSar1a. Structure homology model- whether tissue specificity or functional diversity exists for plant ing and interaction studies revealed that Cys84 is crucial for the Arabidopsis COPII isoforms. So far, only two studies have pointed toward a specific interaction of AtSar1a with AtSec23a, a distinct functional diversity for COPII homologs in Arabidopsis. Genetic Sec23 homolog. Structure modeling and coimmunoprecipitation fur- screening has identified a recessive mis-sense point mutation ther identified a corresponding amino acid, Cys484, on AtSec23a as (R693K) in Sec24A, which induces the formation of ER and Golgi being essential for the specific pair formation. At the cellular level, the membrane clusters and leads to a partial redistribution of Golgi Cys484 mutation affects the distinct function of AtSec23a on vacuolar and secretory markers in the clusters (11, 12). Interestingly, the cargo trafficking. Additionally, dominant-negative AtSar1a affects the expression of Sec24A or YFP-Sec24A complemented the mis- ER export of the transcription factor bZIP28 under ER stress. We sense mutation phenotype but did not occur with Sec24B and have demonstrated a unique plant pair of COPII machinery function Sec24C, indicating the existence of functional diversity among the in ER export and the mechanism underlying the functional diversity Arabidopsis of COPII paralogs in eukaryotes. Sec24 paralogs. More strikingly, transient expression experiments in tobacco protoplasts have shown that AtSar1a coat protein complex II | Sar1 | Sec23 | ER export | functional diversity (At1g09180), which shares a 93% amino acid sequence identity Significance he molecular basis for protein export out of the endoplasmic Treticulum (ER) has been built on the isolation and character- ization of sec mutants that accumulate ER membranes at the non- The underlying mechanisms causing the functional diversity of permissive temperature in yeast (1). The vesicle coat proteins small GTPase Sar1 paralogs in coat protein complex II-mediated responsible for ER-Golgi transport, collectively termed the “coat protein endoplasmic reticulum (ER) export remain elusive in Arabidopsis protein complex II” (COPII), were discovered by combining this higher organisms. contains five Sar1 homologs. In genetic approach with biochemical assays (2). The COPII vesicu- this study, we show that AtSar1a exhibits a distinct localization lating machinery consists of five cytosolic proteins: Sar1, Sec23, and effects on ER cargo export in plants through a unique in- Sec24, Sec13, and Sec31. The small GTPase Sar1 (Secretion-asso- teraction with the COPII coat protein AtSec23a. This specific ciated and RAS superfamily-related protein 1) (3) is first activated pairing is required for the distinct function in ER export under ER stress in Arabidopsis. Our results point to a mechanism un- and recruited to the ER membrane by the guanosine nucleotide derlying the functional diversity of COPII paralogs in eukaryotes. exchange factor Sec12, an ER-localized integral membrane protein (4). Subsequently, the GTPase activating protein (GAP) Sec23, Author contributions: Y.Z., K.-B.W., R.S., and L.J. designed research; Y.Z., K.P.C., B.L., C.M.L., which stimulates the enzymatic activity of Sar1 (5), and the adaptor S.K.L., X.W., Y.C., C.G., and M.L. performed research; Y.Z., C.M.L., and K.-B.W. analyzed data; protein Sec24 (6), are recruited to the ER membrane as a hetero- and Y.Z. and L.J. wrote the paper. dimer by Sar1-GTP to form a prebudding complex (7). This complex The authors declare no conflict of interest. in turn recruits a Sec13/Sec31 heterotetramer, which forms the outer This article is a PNAS Direct Submission. layer of the COPII coat, completing the vesicle formation process. 1To whom correspondence should be addressed. Email: [email protected]. In mammals, COPII transport has diversified due to gene- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. duplication events, creating multiple paralogs of four of the COPII 1073/pnas.1519333112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1519333112 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 with other Arabidopsis Sar1 homologs, exhibited different effects antibody against the endogenous cis-Golgi protein EMP12 and on the secretion of a soluble secretory reporter α-amylase (13). showed that AtSar1c localized at puncta only partially overlapped However, the mechanisms underlying the functional diversity of with EMP12 and was mainly separate from the Golgi apparatus in the AtSar1a in the proteins ER export remain to be elucidated. Arabidopsis root cells (Fig. 1 Aa and B). Similar results were also Here, we report that the AtSar1a (At1g09180) exhibits a distinct obtained with AtSar1c-GFP transgenic tobacco BY-2 cell lines ER exit site localization that is closely associated with the Golgi (Fig. 1 Ca and D). These results are consistent with previous apparatus in plant cells. We further demonstrate that a dominant- studies that showed that AtSar1b only partially overlapped negative mutant of AtSar1a showed distinct effects on the ER with the Golgi apparatus in plant cells (18, 19). Interestingly, export of vacuolar and membrane cargos in Arabidopsis, which is AtSar1a exhibited a distinct localization in both Arabidopsis root determined by a single amino acid substitution from the conserved cells (Fig. 1 Ab and B) and tobacco BY-2 cells (Fig. 1 Cb and D), as tyrosine to cysteine at position 84 on AtSar1a. Homology mod- most puncta labeled by AtSar1a were peri-Golgi localized. To eling and interaction assays have proved that the amino acid Cys84 further confirm the results, we performed superresolution mi- is crucial for the specific interaction of AtSar1a with AtSec23a croscopy [structured illumination microscopy (SIM)] with AtSar1a- (accession no. At4g01810), a distinct Arabidopsis Sec23 homolog. GFP– and AtSar1c-GFP–transfected Arabidopsis protoplasts. Strikingly, protein structure modeling showed that only AtSec23a AtSar1a-labeled puncta were closely associated with the Golgi contains a corresponding amino acid substitution from conserved marker whereas the AtSar1c-GFP positive punctae were largely aspartate to cysteine at position 484, which contacts with Cys84 on separated from the Golgi apparatus (Fig. 1E). The observation on AtSar1a. This specific amino acid Cyc484 on AtSec23a was further
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