Figure 1.
Schematic representation of 2A function and constructs.
(A) Two individual polypeptides can be generated from one transcript using 2A to link the individual genes. (B) 2A peptide sequence and its cleavage site (G/P). (C–L) The constructs used in this paper are described in the following scheme: 2A, 20 amino acid 2A peptide; CFP, enhanced cyan fluorescent protein; YFP, enhanced yellow fluorescent protein; SP, ER signal peptide of CAH1 protein; GS, Golgi targeting signal of N-acetylglucosaminyl transferase I; GUS, β-glucuronidase; RABD2a, Arabidopsis RABD2a GTPase; SAR1, Nicotiana tabacum SAR1p; ARF1, Arabidopsis ADP-ribosylation factor 1; HA, hemagglutinin epitope tag; CAH1, Arabidopsis α-CAH1. All GTPases were cloned as wild type and dominant mutant versions.
Figure 2.
Targeting of 2A constructs and 2A cleavage efficiency.
(A–H) Bright field and confocal images of Arabidopsis protoplasts transiently expressing CFP-2A-RABD2a (A and B), SP-CFP-2A-RABD2a (C and D), GS-CFP-2A-RABD2a (E and F) and CFPGUS-2A-RABD2a (G and H). Bars = 5 µm. (I) Immunoblot analysis of protein extracts from Arabidopsis protoplasts transiently transfected with non-tagged RABD2a (negative control), CFP (positive control), CFP-2A-RABD2a, SP-CFP-2A-RABD2a, GS-CFP-2A-RABD2a and CFPGUS-2A-RABD2a using anti-GFP antiserum. 2A cleaved (*), non-cleaved full-length 2A polyproteins (**) and putative degradation products (−) are indicated. Protein loading was adjusted in order to highlight both cleaved and non-cleaved product for each construct. (J) Cleavage efficiency of the different 2A constructs was estimated from the amounts of cleaved (*) versus non-cleaved (**) products as described in Material and Methods. Error bars show standard error (n = 4, *** = p<0.001).
Figure 3.
RABD2a protein is stable when expressed from 2A constructs.
Immunoblot analysis of protein extracts from Arabidopsis protoplasts transiently transfected with non-tagged RABD2a (positive control), CFP (negative control), CFP-2A-RABD2a, SP-CFP-2A-RABD2a, GS-CFP-2A-RABD2a and CFPGUS-2A-RABD2a using anti-RABD2 antiserum. Native RABD (red arrow, N), overexpressed RABD2a (black arrow, O), 2A derived RABD2a (black arrow, *) and non-cleaved 2A polyprotein (**) are indicated. Low expression and high cleavage efficiency of CFPGUS-2A-RABD2a construct makes no detectable full length CFPGUS-2A-RABD2a band, and only faint band of released RABD2a (*) visible at this exposure time.
Figure 4.
Analysis of activity of the RABD2a protein originating from 2A constructs.
(A and B) Immunoblot analysis using anti-GFP antiserum of secreted (A) and intracellular (B) protein extracts from Arabidopsis protoplasts transiently transfected with (wt) or mutant (N121I) SP-CFP-2A-RABD2a. Upper band (**) corresponds to non-cleaved full length SP-CFP-2A-RABD2a and lower (*) to cleaved SP-CFP-2A polypeptide. (C) The amount of secreted (Secreted) and non-secreted intracellular SP-CFP-2A (Intracellular) were quantified. AU, arbitrary unit, error bars show standard error (n = 4, *** = p<0.001). (D-K) Confocal images of transiently transfected Arabidopsis protoplasts co-expressing wild type (D-G) or mutant (N121I) forms (H-K) of SP-CFP-2A-RABD2a and GS-YFP-2A-RABD2a. CFP channel is shown in green and YFP channel in magenta. Merged images (G and K) show co-localization (white). Bars = 5 µm.
Figure 5.
Analysis of activity of SAR1 and ARF1originating from 2A constructs.
Representative confocal images of Arabidopsis protoplasts transiently expressing GS-CFP-2A-SAR1 (wt) (A and B), GS-CFP-2A-SAR1 (H74L) (C and D); GS-CFP-2A-ARF1 (wt) (E and F), GS-CFP-2A-ARF1 (T31N) (G and H) and GS-CFP-2A-ARF1 (Q71L) (I and J). Bars = 5 µm. (K) Analysis of fluorescent Golgi stacks size in Arabidopsis protoplasts transiently expressing GS-CFP-2A-ARF1 (wt) or mutant (Q71L) forms. Error bars show standard error (n = 14, *** = p<0.001).
Figure 6.
Effect of mutant forms of small GTPases on trafficking of HACAH1-2A.
Immunoblot analysis using anti-HA antiserum of protein extracts from Arabidopsis protoplasts (A, B and C) and Nicotiana benthamiana leaves (D and E) expressing HACAH1-2A-RABD2a (wt) or mutant (N121I) form (A and D); HACAH1-2A-SAR1 (wt) or mutant (H74L) form (B); HACAH1-2A-ARF1 (wt) or mutant (T31N) and (Q71L) forms (C and E). Full length non-cleaved HACAH1-2A-RABD2A/SAR1/ARF1 (**), 2A cleaved HACAH1-2A (*), Endo H resistant (black triangle) and susceptible (white triangle) forms of HACAH1-2A are indicated. (F and G) The ratio of resistant (black triangle) to total HACAH1-2A (white+black triangle) cleaved forms (*) was calculated for Arabidopsis protoplasts (F) and N. benthamiana leaves (G). Error bars show standard error (n = 4, ** = p<0.01, *** = p<0.001).