Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway.
Cardosin A is the major vacuolar aspartic proteinase (APs) (E.C.3.4.23) in pistils of Cynara cardunculus L. (cardoon). Plant APs carry a unique domain, the plant-specific-insert (PSI), and a pro-segment which are separated from the catalytic domains during maturation but the sequence and location of processing steps for cardosins have not been established. Here transient expression in tobacco and inducible expression in Arabidopsis indicate that processing of cardosin A is conserved in heterologous species. Pulse chase analysis in tobacco protoplasts indicated that cleavage at the carboxy-terminus of the PSI could generate a short-lived 50 kDa intermediate which was converted to a more stable 35 kDa intermediate by removal of the PSI. Processing intermediates detected immunologically in tobacco leaves and Arabidopsis seedlings confirmed that cleavage at the amino-terminus of the PSI either preceded or followed quickly after cleavage at its carboxy-terminus. Thus removal of PSI preceded the loss of the prosegment in contrast to the well-characterised barley AP, phytepsin. PreprocardosinA acquired a complex glycan and its processing was inhibited by brefeldin A and dominant-inhibitory AtSAR1 or AtRAB-D2(a )mutants indicating that it was transported via the Golgi and that processing followed ER export. The 35 kDa intermediate was present in the cell wall and protoplast culture medium as well as the vacuole but the 31 kDa mature subunit, lacking the amino-terminal prosegment, was detected only in the vacuole. Thus maturation appears to occur only after sorting from the trans-Golgi to the vacuole. Processing or transport of cardosin A was apparently slower in tobacco protoplasts than in whole cells.
Duarte
Patrícia
P
Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, Porto, Portugal. pduarte@ibmc.up.pt
Pissarra
José
J
Moore
Ian
I
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031942:Cynara / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D035264:Flowers / Q000201:enzymology*; D006056:Golgi Apparatus / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism*; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology; D014617:Vacuoles / Q000201:enzymology*
D029681:Arabidopsis Proteins; D007527:Isoenzymes; D010940:Plant Proteins; D011134:Polysaccharides; D020033:Protein Isoforms; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus