The role of the N region in signal sequence and signal-anchor function.
To determine the role of sequences other than the hydrophobic core in mediating signal sequence function, we examined the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that neither the N nor the C region of the preprolactin signal sequence is necessary for translocation. However, insertion of sequences with either a net charge of +2.5 or -6.0 between the N region and the hydrophobic core of the signal converted it into a signal-anchor. The topologies adopted (types I and II, respectively) were opposite those predicted from the distribution of charges surrounding the hydrophobic core of the signals. When these mutant signals were located in the interior of an otherwise secreted protein, both sequences functioned as stop-transfer sequences. Related mutations were assayed in fusion proteins in which the IgM transmembrane domain functioned as an amino-terminal signal-anchor. For these molecules, the distribution of charged residues surrounding the hydrophobic core had no influence on the topology adopted. Our results suggest that features other than simple charge distribution play an important role in determining membrane topology in vitro.
Andrews
D W
DW
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Young
J C
JC
Mirels
L F
LF
Czarnota
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D007075:Immunoglobulin M / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D002679:Pan troglodytes; D010957:Plasmids; D011388:Prolactin / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D007075:Immunoglobulin M; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011388:Prolactin