{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T00:43:45Z","timestamp":1772585025906,"version":"3.50.1"},"reference-count":50,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2019,8,30]],"date-time":"2019-08-30T00:00:00Z","timestamp":1567123200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","award":["PTDC\/BIA-FBT\/32013\/2017"],"award-info":[{"award-number":["PTDC\/BIA-FBT\/32013\/2017"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.<\/jats:p>","DOI":"10.3390\/plants8090312","type":"journal-article","created":{"date-parts":[[2019,8,30]],"date-time":"2019-08-30T10:31:17Z","timestamp":1567161077000},"page":"312","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert"],"prefix":"10.3390","volume":"8","author":[{"given":"Vanessa","family":"Vieira","sequence":"first","affiliation":[{"name":"Faculdade de Ci\u00eancias da Universidade do Porto, Rua do Campo Alegre, s\/n\u00ba, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1022-3908","authenticated-orcid":false,"given":"Bruno","family":"Peixoto","sequence":"additional","affiliation":[{"name":"Faculdade de Ci\u00eancias da Universidade do Porto, Rua do Campo Alegre, s\/n\u00ba, 4169-007 Porto, Portugal"},{"name":"Instituto Gulbenkian de Ci\u00eancia, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal"}]},{"given":"M\u00f3nica","family":"Costa","sequence":"additional","affiliation":[{"name":"Faculdade de Ci\u00eancias da Universidade do Porto, Rua do Campo Alegre, s\/n\u00ba, 4169-007 Porto, Portugal"},{"name":"Instituto Gulbenkian de Ci\u00eancia, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal"}]},{"given":"Susana","family":"Pereira","sequence":"additional","affiliation":[{"name":"Faculdade de Ci\u00eancias da Universidade do Porto, Rua do Campo Alegre, s\/n\u00ba, 4169-007 Porto, Portugal"},{"name":"GreenUPorto-Sustainable Agrifood Production Research Center, Campus de Vair\u00e3o, Rua Padre Armando Quintas 7, 4485-661 Vila do Conde, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9489-9904","authenticated-orcid":false,"given":"Jos\u00e9","family":"Pissarra","sequence":"additional","affiliation":[{"name":"Faculdade de Ci\u00eancias da Universidade do Porto, Rua do Campo Alegre, s\/n\u00ba, 4169-007 Porto, Portugal"},{"name":"GreenUPorto-Sustainable Agrifood Production Research Center, Campus de Vair\u00e3o, Rua Padre Armando Quintas 7, 4485-661 Vila do Conde, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8050-5102","authenticated-orcid":false,"given":"Cl\u00e1udia","family":"Pereira","sequence":"additional","affiliation":[{"name":"Faculdade de Ci\u00eancias da Universidade do Porto, Rua do Campo Alegre, s\/n\u00ba, 4169-007 Porto, Portugal"},{"name":"GreenUPorto-Sustainable Agrifood Production Research Center, Campus de Vair\u00e3o, Rua Padre Armando Quintas 7, 4485-661 Vila do Conde, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2019,8,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1104\/pp.107.102863","article-title":"Molecular dissection of endosomal compartments in plants","volume":"145","author":"Mettbach","year":"2007","journal-title":"Plant Physiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1482","DOI":"10.1104\/pp.108.120105","article-title":"The endosomal system of plants: Charting new and familiar territories","volume":"147","author":"Robinson","year":"2008","journal-title":"Plant Physiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1016\/j.pbi.2009.08.004","article-title":"Plant vacuoles: Where did they come from and where are they heading?","volume":"12","author":"Zouhar","year":"2009","journal-title":"Curr. Opin. Plant Biol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Neuhaus, J.-M., and Rogers, J.C. (1998). Sorting of proteins to vacuoles in plant cells. Protein Trafficking in Plant Cells, Springer.","DOI":"10.1007\/978-94-011-5298-3_7"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"7611","DOI":"10.3390\/ijms15057611","article-title":"Delivering of proteins to the plant vacuole\u2014An update","volume":"15","author":"Pereira","year":"2014","journal-title":"Int. J. Mol. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1016\/j.tplants.2005.05.001","article-title":"Sorting of proteins to storage vacuoles: How many mechanisms?","volume":"10","author":"Vitale","year":"2005","journal-title":"Trends Plant Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"782","DOI":"10.1111\/j.1365-313X.2009.04113.x","article-title":"An engineered C-terminal disulfide bond can partially replace the phaseolin vacuolar sorting signal","volume":"61","author":"Pompa","year":"2010","journal-title":"Plant J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.plaphy.2013.10.010","article-title":"Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting","volume":"73","author":"Stigliano","year":"2013","journal-title":"Plant Physiol. Biochem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1111\/tpj.12274","article-title":"Cardosin A contains two vacuolar sorting signals using different vacuolar routes in tobacco epidermal cells","volume":"76","author":"Pereira","year":"2013","journal-title":"Plant J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1111\/j.1365-313X.2010.04421.x","article-title":"Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco","volume":"65","author":"Lenucci","year":"2011","journal-title":"Plant J."},{"key":"ref_11","first-page":"79","article-title":"Trafficking routes to the plant vacuole: Connecting alternative and classical pathways","volume":"69","author":"Barozzi","year":"2017","journal-title":"J. Exp. Bot."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e25129","DOI":"10.4161\/psb.25129","article-title":"Unconventional pathways of secretory plant proteins from the endoplasmic reticulum to the vacuole bypassing the Golgi complex","volume":"8","author":"Bellucci","year":"2013","journal-title":"Plant Signal. Behav."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1046\/j.1432-1327.1998.2550133.x","article-title":"Identification and proteolytic processing of procardosin A","volume":"255","author":"Cortes","year":"1998","journal-title":"Eur. J. Biochem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1023\/A:1010675015318","article-title":"Molecular cloning and characterization of cDNA encoding cardosin B, an aspartic proteinase accumulating extracellularly in the transmitting tissue of Cynara cardunculus L.","volume":"45","author":"Vieira","year":"2001","journal-title":"Plant Mol. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1007\/s00709-008-0288-9","article-title":"Cardosins in postembryonic development of cardoon: Towards an elucidation of the biological function of plant aspartic proteinases","volume":"232","author":"Pereira","year":"2008","journal-title":"Protoplasma"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1255","DOI":"10.1007\/s00425-008-0697-1","article-title":"Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway","volume":"227","author":"Duarte","year":"2008","journal-title":"Planta"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1517","DOI":"10.1007\/s00425-010-1276-9","article-title":"Dissecting cardosin B trafficking pathways in heterologous systems","volume":"232","author":"Costa","year":"2010","journal-title":"Planta"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.plantsci.2009.11.008","article-title":"Characterization of aspartic proteinases in C. cardunculus L. callus tissue for its prospective transformation","volume":"178","author":"Oliveira","year":"2010","journal-title":"Plant Sci."},{"key":"ref_19","first-page":"274","article-title":"From flower to seed germination in Cynara cardunculus: A role for aspartic proteinases","volume":"1","author":"Pissarra","year":"2007","journal-title":"Int. J. Plant Dev. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2059","DOI":"10.1093\/jxb\/erz034","article-title":"Atypical and nucellin-like aspartic proteases: Emerging players in plant developmental processes and stress responses","volume":"70","author":"Soares","year":"2019","journal-title":"J. Exp. Bot."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2067","DOI":"10.1111\/j.1432-1033.2004.04136.x","article-title":"Structure and function of plant aspartic proteinases","volume":"271","author":"Faro","year":"2004","journal-title":"Eur. J. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1016\/j.bbamem.2013.08.004","article-title":"Possible mechanism of structural transformations induced by StAsp-PSI in lipid membranes","volume":"1838","author":"Daleo","year":"2014","journal-title":"Biochim. Biophys. Acta-Biomembr."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"38190","DOI":"10.1074\/jbc.M006093200","article-title":"The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage","volume":"275","author":"Egas","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.phytochem.2018.02.004","article-title":"Transgenic expression of plant-specific insert of potato aspartic proteases (StAP-PSI) confers enhanced resistance to Botrytis cinerea in Arabidopsis thaliana","volume":"149","author":"Frey","year":"2018","journal-title":"Phytochemistry"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1016\/j.peptides.2010.02.001","article-title":"The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens","volume":"31","author":"Mendieta","year":"2010","journal-title":"Peptides"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"De Moura, D.C., Bryksa, B.C., and Yada, R.Y. (2014). In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0104315"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1128\/AEM.03151-13","article-title":"Establishing the yeast kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin","volume":"80","author":"Curto","year":"2014","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1016\/j.jplph.2005.08.007","article-title":"Plant-specific insertions in the soybean aspartic proteinases, soyAP1 and soyAP2, perform different functions of vacuolar targeting","volume":"163","author":"Terauchi","year":"2006","journal-title":"J. Plant Physiol."},{"key":"ref_29","first-page":"2021","article-title":"A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum","volume":"13","author":"Hadlington","year":"2007","journal-title":"Plant Cell"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1046\/j.1365-313X.1998.00208.x","article-title":"Stacks on tracks: The plant Golgi apparatus traffics on an actin\/ER network","volume":"15","author":"Boevink","year":"1998","journal-title":"Plant J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1046\/j.0960-7412.2002.01252.x","article-title":"Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks","volume":"29","author":"Evins","year":"2002","journal-title":"Plant J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1753","DOI":"10.1105\/tpc.022673","article-title":"Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells","volume":"16","author":"Snapp","year":"2004","journal-title":"Plant Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1093\/jxb\/erp315","article-title":"Golgi membrane dynamics after induction of a dominant-negative mutant Sar1 GTPase in tobacco","volume":"61","author":"Osterrieder","year":"2010","journal-title":"J. Exp. Bot."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1242\/jcs.103.4.1153","article-title":"Redistribution of a Golgi glycoprotein in plant cells treated with Brefeldin A","volume":"103","author":"Hawes","year":"1992","journal-title":"J. Cell Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3081","DOI":"10.1105\/tpc.105.034900","article-title":"Diacidic motifs influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells","volume":"17","author":"Hanton","year":"2005","journal-title":"Plant Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2201","DOI":"10.1105\/tpc.12.11.2201","article-title":"A rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants","volume":"12","author":"Batoko","year":"2000","journal-title":"Plant Cell"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1034","DOI":"10.1111\/j.1600-0854.2009.00930.x","article-title":"Fluorescence lifetime imaging of interactions between Golgi tethering factors and small GTPASES in plants","volume":"10","author":"Osterrieder","year":"2009","journal-title":"Traffic"},{"key":"ref_38","unstructured":"Gupta, R., Jung, E., and Brunak, S. (2003, October 29). \u201cPrediction of N-glycosylation Sites in Human Proteins.\u201d In Preparation. Available online: http:\/\/www.cbs.dtu.dk\/services\/NetNGlyc\/."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1007\/s00425-003-1179-0","article-title":"Characterization of the genes for two soybean aspartic proteinases and analysis of their different tissue-dependent expression","volume":"218","author":"Terauchi","year":"2004","journal-title":"Planta"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1007\/s00299-009-0799-7","article-title":"Expression of a glycosylated GFP as a bivalent reporter in exocytosis","volume":"29","author":"Paris","year":"2010","journal-title":"Plant Cell Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1093\/jxb\/49.326.1463","article-title":"The protein N-glycosylation in plants","volume":"49","author":"Rayon","year":"1998","journal-title":"J. Exp. Bot."},{"key":"ref_42","first-page":"301","article-title":"Role of propeptide glycan in post-translational processing and transport of barley lectin to vacuoles in transgenic tobacco","volume":"2","author":"Wilkins","year":"1990","journal-title":"Plant Cell"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1093\/jexbot\/52.358.911","article-title":"Deglycosylation is necessary but not sufficient for activation of proconcanavalin A","volume":"52","author":"Ramis","year":"2001","journal-title":"J. Exp. Bot."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/S1360-1385(99)01389-8","article-title":"What do proteins need to reach different vacuoles?","volume":"4","author":"Vitale","year":"1999","journal-title":"Trends Plant Sci."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Occhialini, A., Gouzerh, G., Di Sansebastiano, G.P., and Neuhaus, J.M. (2016). Dimerization of the vacuolar receptors AtRMR1 and -2 from Arabidopsis thaliana contributes to their localization in the trans-Golgi network. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17101661"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Pompa, A., De Marchis, F., Pallotta, M.T., Benitez-Alfonso, Y., Jones, A., Schipper, K., Moreau, K., \u017d\u00e1rsk\u00fd, V., Di Sansebastiano, G.P., and Bellucci, M. (2017). Unconventional transport routes of soluble and membrane proteins and their role in developmental biology. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18040703"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/jxb\/erx435","article-title":"Protein and membrane trafficking routes in plants: Conventional or unconventional?","volume":"69","author":"Goring","year":"2017","journal-title":"J. Exp. Bot."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3947","DOI":"10.1093\/emboj\/18.14.3947","article-title":"Crystal structure of plant aspartic proteinase prophytepsin: Inactivation and vacuolar targeting","volume":"18","author":"Kervinen","year":"1999","journal-title":"EMBO J."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2019","DOI":"10.1038\/nprot.2006.286","article-title":"Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants","volume":"1","author":"Sparkes","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1104\/pp.103.027979","article-title":"Breakthrough Technologies A gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta","volume":"133","author":"Curtis","year":"2003","journal-title":"Plant Physiol."}],"container-title":["Plants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2223-7747\/8\/9\/312\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:15:15Z","timestamp":1760188515000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2223-7747\/8\/9\/312"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,8,30]]},"references-count":50,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2019,9]]}},"alternative-id":["plants8090312"],"URL":"https:\/\/doi.org\/10.3390\/plants8090312","relation":{"has-preprint":[{"id-type":"doi","id":"10.20944\/preprints201908.0027.v1","asserted-by":"object"}]},"ISSN":["2223-7747"],"issn-type":[{"value":"2223-7747","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,8,30]]}}}