Fig 1. RPN-10 is broadly expressed and contributes to UPS activity. (A) The expression of an RPN-10::GFP fusion protein from a fosmid-basedtransgene shows broad expression in the cytoplasm and nuclei (arrow) of multiple tissues including the intestine, pharynx, hypodermis, and germline. The GFP signal is produced from the
rpn-10 gene because it can be effectively silenced via treatment of the worms with
rpn-10 RNAi (B). (C) Quantification of GFP expression in digital images captured as in Panel B via the use of ImageJ. *** represents p = 0.001 by t-test. (D) Structure of the full-length RPN-10 protein showing the location of the N-terminal Von Willebrand factor A domain (VWA), the location of the highly conserved Asp-Asn-Ser-Glu (DNSE)sequence that is essential for the binding of RPN-10 to the 19S proteasome subunit, and the two C-terminal ubiquitin interacting domains (UID). The dotted line indicates the coding sequence regions deleted in the
rpn-10(
ok1865) allele, and this line extends beyond the N-terminus of the RPN-10 protein to highlight the extension of the deletion into the 5' UTR of the
rpn-10 gene. (E) The
rpn-10(
ok1865) mutation disrupts UPS function as shown by the selective accumulation of a UbV::GFP fusion protein in the intestine of the mutant but not wild-type animals. In contrast, the
rpn-10 mutation has no effect on the expression of mCherry driven by the same promoter from a separate transgene in the animals. (F) Quantification of GFP expression from digital images captured as in Panel E. *** represents p<0.001 by t-test. (G) The accumulated UbV::GFP fusion protein is localized in the intestine except for the three proximal cells (arrow). (H) In addition to accumulating the UbV::GFP fusion protein, the
rpn-10 mutant induces the expression of the
aip-1p::GFP reporter gene in multiple tissues along with the intestine. (I) Quantification of GFP expression from digital images captured as in Panel H. *** represents p<0.001 by t-test.