FIGURE 9.

The lower GOAT band is a protein with N terminus Met-56. A, MALDI-TOF of intact, purified tag-cleaved mouse GOAT. Expected molecular weights (MW) with the indicated start codons are inset (estimated S.E. for these masses is ±50 Da). B, adding N-terminal sequence shifts the upper band but not the lower band in mouse GOAT. *, all constructs contain a C-terminal 3xFLAG tag. N-IBV1 and N-IBV2 are described under “Experimental Procedures”; 3xV5Glyc is V5-Glyc-V5-Glyc-V5; V5Glyc is V5-Glyc-V5; Glyc-V5 lacks the first V5 epitope of V5Glyc. C, anti-FLAG immunoblot of SF9 microsomes of full-length and helix-deleted constructs. ΔN-H1 construct lacks the first helix, ΔN-H1-2 lacks the first two helices, etc. The start codon for ΔN-H1-2 is Met-56; for ΔN-H1-3 and ΔN-H1-4, a Met was installed in place of residues 81 and 109, respectively, as the first residue of the construct. All deletion mutants have a C-terminal TEV-3xFLAG. Human GOAT proteins are labeled 46A and 46T because this position was expressed as either the conserved Ala or the recorded coding SNP Thr. 3-Fold more human GOAT-expressing microsomes were loaded than mouse GOAT-expressing microsomes, due to lower expression. D, mutation of Met-56 removes the lower band from mouse GOAT. Met-56 was mutated to alanine or isoleucine for the indicated constructs, and all were expressed in HeLa cells. Expression levels are reduced by 5–10-fold for mutant constructs (not shown); here, 4 μg of total protein was loaded for Met constructs, and 20 μg was loaded for Ala and Ile constructs. E, microsomal activity assay of TEV-3xFLAG tagged GOAT constructs in C. Each bar represents an average of duplicates; error bars, S.D.