Figure 5.
The G-domain and RG-domain of Grn1p are required for growth. (A) The growth of all the indicated strains: WT (YNB544), Δgrn1 (YNB546), ΔRG (YNB568), ΔG3 (YNB956), ΔG1 (YNB545), ΔG4 (YNB611), ΔG5 (YNB566), and ΔCC (YNB567) was determined. Strains were struck for single colonies on EMM-leu plates with (nmt1 OFF) or without 15 μM thiamine (nmt1 ON). (B) The total proteins of all the strains were isolated and processed by Western using anti-GFP antibody. (C) GFP, Grn1:GFP, ΔG5-Grn1:GFP, ΔG4-Grn1:GFP, ΔG1-Grn1:GFP, ΔG3-Grn1:GFP, ΔRG-Grn1:GFP, from pBNB340, pBNB338, pBNB335, pBNB336, pBNB337, pBNB417, and pBNB339, respectively (Table 2), were transcribed and translated in the presence of l-[35S]methionine using a TNT-coupled Reticulocyte Lysate System (Promega, Madison, WI) according to the manufacturer's instructions. One microliter of each of product was analyzed on a 12% SDS-PAGE gel and exposed to x-ray film.