Figure 5. GOLM1-MAK10 leads to the translation of a secreted fusion protein.

(A) Schematic of GOLM1-MAK10 chimera. The chimera of GOLM1-MAK10 could result in the substitution of the last 25 amino acids of GOLM1 by 26 new amino acids with the 3' UTR from MAK10. In the pictogram, coding exons are represented by blocks connected by horizontal lines representing introns. The 5′- and 3′-UTR are represented by shorter blocks. Arrows indicate direction of transcription of parental genes. Dashed lines indicate the boundaries of the sequence contributing to chimeric RNAs. Splice junctions are shown as uppercase letters. The sequences in black are part of the introns removed in the formation of the chimeric RNAs. Note that a discernible 5' splice site and a 3' splice site are present at the RNA junction. (B) Chimera RNA GOLM1-MAK10 is predicted to translate into a fusion protein with the majority of amino acid sequence of GOLM1 retained, and has a near identical size to the GOLM1 parental protein. GOLM1-MAK10 would retain the signal peptide region and the transmembrane domain that are required for secretion. The coiled-coil domain required for the dimerization of GOLM1 also remains unchanged. (C) GOLM1-MAK10 construct with FLAG tag was expressed under the control of the CMV promoter in HEK 293T cells. Fusion protein is efficiently translated and secreted in the media as dimers. For FLAG western, 0.4% of the total cell extract and 0.4% of the total media were loaded. For Actin western, equal amounts of extracts were loaded. (D) Iodoacetamide treatment, which reduces the disulfide bonds, partially converted dimers into monomers. The expected size of the monomer is 44 kD. However, we observe bands at roughly 60 kD for the monomer, suggesting that they are heavily glycosylated. This is consistent with previous observations of GOLM1.