Figure 1.

MCF autoproteolytic cleavage is induced by ARF GTPases.

A. dt-cMCF^CA was immunoprecipitated (IP) from HEK293T cell lysate using anti-HA antibody. Box indicates band excised for mass spectrometry peptide sequencing. B. Amino acid sequence alignment of major isoforms of ARFs with boxes indicating identified peptides. Peptides marked 6 and 7 are unique to ARF4 and ARF6, respectively. C. Immunoblot showing endogenous ARF1 pulls-down with dt-cMCF^CA recovered from whole cell lysate as detected by anti-ARF1 antibody and anti-HA. Replicate pull-downs of independent transfections can be found in Supplemental Fig. 1. D. Purified GST-Δ17 ARF1 was incubated with either MCF, MCF^CA, cMCF, or cMCF^CA at 37°C. Western blot on samples following anti-GST IP using anti-MCF (green bands) and anti-ARF1 (red bands). showing catalytically active and inactive flMCF and cMCF (green bands) can bind ARF1(red bands) in vitro. E. Recombinant flMCF and flMCF^CA incubated with ΔARF1 indicate induced autoprocessing is dependent on MCF catalytic active site. Arrow indicates cleaved MCF band. F. Auto-cleavage induced by purified ARF isoforms incubated with MCF for 24 hours. G, H. Autoprocessing of flMCF at 37°C for time indicated induced by full-length ARF1 or ARF3 (F) or with ARF1 pre-loaded with GDP or non-hydrolyzable GTP (GppNHp) (G). Representative gels (n>3), with percent cleaved MCF was determined by densitometry of bands on Coomassie stained gels (%=(cMCF/(cMCF + flMCF)*100)) from three independent reactions.