Fig. 4: Detection of ex-proteasomes in the apoplast by MS and immunoblot assays.

a Identification of proteasome subunits by LC-MS/MS analysis of the APF, APFp, and CL. Listed are all Arabidopsis proteasome subunit isoforms; those detected in the APF, APFp, and CL are marked in red, orange, and green type, respectively. The catalog was separated by the heptameric CP α and β rings, and the RP Lid and Base subcomplexes (see³⁵ for reference). Whereas most, if not all, of the 14 CP subunits were detected in the APF/APFp, a few of the 19 RP subunits were not (RPN10, RPN13, and RPN14/SEM1). b Immunoblot detection of ex-proteasome subunits in the APFp isolated from wild-type analyzed alongside a dilution series of the CL. Immunoblot analysis with anti-cFPB antibodies was included as a control. For a second control, the APFp was isolated from a line expressing a GFP-tagged subunit of the ribosome large-subunit protein RPL18 and immunoblotted with anti-GFP antibodies. c Relative abundance of the 26S proteasome and the CP and RP subcomplexes in the CL, APF, and APFp fractions as determined by MS/MS. The values were calculated by combining normalized MS1 data for all subunits of the indicated complexes. d Specific activity per mg protein for proteasomes in the APFp and CL based on hydrolysis of the CP substrate Suc-LLVY-AMC. e Immunoblotting for CP and RP subunits using equivalent amounts of proteolytic activity for the CL and APFp as determined in panel (d). f Immunoblot detection of Ub and Ub conjugates in the CL and APFp using anti-Ub antibodies. Ub conjugates and free Ub are located by the bracket and arrowhead, respectively. Bars in panels (c) and (d) reflect the mean (±SD) of four and three technical replicates, respectively. Individual data points are included.