A zein signal sequence functions as a signal-anchor when fused to maize alcohol dehydrogenase.
A chimeric gene, preZad, was constructed encoding a zein signal sequence fused precisely to the amino terminus of maize alcohol dehydrogenase 1. Translocation and processing of this chimeric preZad protein were assayed in vitro using a rabbit reticulocyte lysate translation system supplemented with canine pancreatic microsomes. PreZad was cotranslationally translocated across the vesicular membranes. Unexpectedly, the signal sequence was not removed although a suitable cleavage site was preserved and presented within the vesicle lumen. Failure to cleave the signal sequence was apparently not due to the lack of a beta-turn near the processing site. When a beta-turn was introduced near the cleavage site through site-directed mutagenesis, no processing was observed. PreZad was not solubilized by alkaline treatment of the microsomes, indicating an integral membrane association. Resistance to proteolysis, in the absence of detergent, indicates that preZad is associated with the membranes in a type II orientation (C-terminus in and N-terminus outside the vesicles). Analysis of truncated versions of preZad showed that it is the uncleaved signal sequence that functions as a signal-anchor. Changing the ratio of net charge flanking the signal sequence to less than 1 (N-terminal:C-terminal) did not alter the type II membrane orientation, as would have been predicted by the 'positive-in rule'. Our results provide additional insight into the role of the passenger protein and signal sequence-flanking regions in recognition of a signal peptidase processing site, and the orientation of insertion of a signal-anchor sequence into the endoplasmic reticulum membrane.
Shatters
R G
RG
Seed Biosynthesis Research Unit, USDA, Agriculture Research Service, National Center for Agricultural Utilization Research, Peoria, IL.
Miernyk
J A
JA
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002678:Chimera; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics* / Q000502:physiology; D012313:RNA / Q000096:biosynthesis; D014158:Transcription, Genetic; D014178:Translocation, Genetic; D003313:Zea mays / Q000737:chemistry; D015028:Zein / Q000235:genetics*
D011498:Protein Precursors; D021382:Protein Sorting Signals; D012313:RNA; D015028:Zein; D000426:Alcohol Dehydrogenase