Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY.
SecY is an integral plasma-membrane protein of Escherichia coli which is essential for the export of periplasmic and outer-membrane proteins containing cleavable signal sequences. We have synthesized SecY in vitro using an E. coli transcription/translation system. In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside-out plasma-membrane vesicles (INV) had been added cotranslationally. Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment. In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to beta-lactamase and lambda receptor. When SecY-deprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the lambda receptor were partially restored. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the secY gene suppresses signal-sequence mutations of the lambda receptor in vitro. Collectively, our results demonstrate that SecY, while functioning as a membrane-located receptor for precursors of exported proteins, appears to be virtually independent of pre-existing SecY for its own membrane integration.
Swidersky
U E
UE
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Rienhöfer-Schweer
A
A
Werner
P K
PK
Ernst
F
F
Benson
S A
SA
Hoffschulte
H K
HK
Müller
M
M
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000378:metabolism; D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D016153:Genes, Suppressor; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D018272:Porins; D011233:Precipitin Tests; D021382:Protein Sorting Signals / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism; D000069816:SEC Translocation Channels; D014357:Trypsin / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018272:Porins; D021382:Protein Sorting Signals; D011991:Receptors, Virus; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; C016532:maltoporins; D014357:Trypsin