Mutation sites were chosen based on the consensus location of the transmembrane domain predicted by the different membrane topology algorithms shown in Table 1 . The results from this analysis showed that the various transmembrane prediction algorithms delineated transmembrane domains with variable N (G424 to I431) and C (Y445 to S449) termini and different numbers of potential GXXXG motifs. All of the predicted transmembrane domains lacked the terminal GLSE predicted by Kyte Doolittle and Hopp-Wood (Table 1 ) and thus did not include the G420-LSE-G424 motif. Ten of the 12 predicted transmembrane domains included G428, strongly suggesting the presence of the two motifs centered on G432 (G428-IVI-G432 and G432-SVA-G436). Although G424 was present in only 3 of 12 predicted TM, G424's location at the N-terminus of a tandem array of three classic GXXXG motifs caused us to consider that the G424-AIA-G432 sequence might function as a dimerization motif when all the other GXXXG motifs were disrupted by G428L and G436L mutations. With regard to the C terminus, all of the predicted transmembrane domains contained Y445 and 7 of 12 contained Y448, leading us to conclude with reasonable certainty that Y448 was within the transmembrane domain or at the very least, at the interface between the transmembrane and cytoplasmic domains. Taking all of these considerations into account, we arrived at the transmembrane sequence proposed in Table 2 . The brackets denote the uncertainty with regard to G424 and Y448. In recognition of the variability in the predicted N-terminus and the uncertainty regarding the functionality of the G424-AIA-G428 motif, we introduced a G424L mutation to disrupt this motif and a G432L mutation to knock out both the G428-IVI-G432 and the G432-SVA-G436 motifs. Disruption of all three of the possible GXXXG motifs was accomplished by introducing both G424L and G432L mutations. To disrupt tyrosine mediated interactions, Y445 and Y448 were replaced with V, the choice of a tyrosine to valine mutation being based on the following considerations: 1) valine lacks a hydroxy group; 2) valine does not have an aromatic side chain; 3) valine is non-polar and thus should not disrupt the alpha helix or alter transport to the plasma membrane.