Although previous studies have shown an initial 'head-first', and hence inverted, insertion of signal-anchor sequences [ 6, 22], the suggestion that TM1 of TASK-1 can assume a completely inverted topology was nevertheless unanticipated.
The replica signal is called a cyclic prefix (CP) because the insertion of that signal resembles the cyclic shift process to the right direction.
The fact that the amount of SP-PrP increased in response to CAM741, which interferes with the correct insertion of the signal peptide into the translocon [36], [37], suggests that a post-targeting interaction between the signal and the Sec61 channel is primarily involved in PrP translocation, a conclusion also emerging from other studies [18], [42].
CAM741, a cyclopeptolide that inhibits co-translational translocation by interfering with the correct insertion of the signal peptide into the translocon [36], [37], increased the amount of SP-PrP in hippocampal and cortical neurons (Fig. 6A, compare lanes 4 and 6, and data not shown).
Insertion of a signal peptide or transmembrane domain into this site would further open the lateral gate, presumably destabilizing the plug.
It is important to emphasize that the addition of GMP-PNP does not prevent targeting, because GTP hydrolysis is not required for the insertion of the signal peptide into the translocon but rather for the dissociation of the SRP-SR complex [ 20].
To this end, several strategies have been employed: (i) multiple promoters fused to the genes' open reading frames (ORFs); (ii) insertion of splicing signals between genes; fusion of genes whose expressions are driven by a single promoter; (iii) insertion of proteolytic cleavage sites between genes; and (iv) insertion of internal ribosomal entry sites (IRESs) between genes [1], [2].
Fortunately, we discovered that insertion of all signaling elements studied here at −60 resulted in a marked diminution of basal activity.
Correct insertion of the signaling sentinels into the Rosa26 locus was ascertained by PCR and restores the Rosa26 locus from +81 to the downstream insertion of the loxP2272 site at +1 kb.
Our data strongly support previous studies [ 6, 22], both of which provide compelling evidence for a pathway involving the 'head-first' insertion of model signal-anchor sequences followed by their subsequent inversion to achieve their native type II topology (N-terminus cytosolic).
