Figure 1
An extra N-terminal region contained in human ACAT1 56-kDa isoform is encoded by a sequence present in recombinant Ampr-plasmids. (A-C) Western blot analysis of the expression of WT and mutant ACAT1 constructs. Constructs (numbers 1-23) containing the entire ACAT1 (A), partial ACAT1 (B, C) or Rluc (C) were transfected separately into AC29 cells. Western blot analyses were performed with anti-ACAT1 or anti-Rluc antibodies. Filled circle, GGC1274-1276 codon; hollow circle, ATG1397-1399 codon; and underlined characters, mutated nucleotides. (D) Calculated molecular weights (MW) of proteins initiated from the GGC1274-1276, ATG1274-1276 and ATG1397-1399 codons. ACAT1-50NT, 130 amino acids encoded by the sequence from ATG1397-1399 to GAT1784-1786. ACAT1-56uNT, 41 amino acids upstream of ACAT1-50NT. Filled circle, glycine; hollow circle, methionine; and aa, amino acids. (E) Purification and 2-D electrophoresis of the 26-kDa ACAT1 protein. Construct pNTF-number 10 (B) was expressed in AC29 cells to generate the 26-kDa ACAT1 protein. The proteins were purified with ANTI-FLAG M2 Affinity Gel and separated by 2-D electrophoresis. Arrows indicate spots of different 26-kDa proteins designated as P1, P2 and P3. PCMV, CMV promoter (black arrow); ori, ColE1 origin (gray arrow). (F) MALDI-TOF MS analysis of the purified 26-kDa proteins. Mass spectra are recorded in the positive mode and listed for the purified 26-kDa protein P2. (G) The N-terminal region of the purified P2 protein and its origin. The 15 amino acids (bold) were determined by the N-terminal sequencing of the purified P2 protein; they are encoded by the underlined sequence that matches the sequence of asAmp derived from recombinant plasmids (asAmp, tessellated box). The amino acid sequence of asAmp-encoding p88 protein (asAmp-P88) and the region of a recombined cryptic promoter are shown. (H) Schematic representation of human ACAT1-56NT region. Italics indicate the N-terminal 15 amino acids of the purified P2 protein. Asterisks indicate modifications by iodoacetamide on cysteines. Trypsin cleavage sites are indicated by bold characters and calculated MWs of representative peptides (underlined) after trypsin cleavage are shown. ACAT1-56eNT, 43 or 46 amino acids from the asAmp-P88. (I) Activity of a recombined cryptic promoter upstream of the asAmp ORF region. Reporter plasmids containing the luciferase gene under the control of various-sized upstream sequences of the asAmp ORF region were transfected into HEK293 cells, and their relative luciferase activities were determined. Means and SDs are shown (n = 3). Value with the promoter-less plasmid is defined as 1.0.
