Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY

Abstract

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 p-lactamase and , I receptor. When SecYdeprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the , I 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 sec Y gene suppresses signal-sequence mutations of the , I 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.

In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways ofEscherichia coli

“…Instead, the SecY translation products were subfractionated on a two-step sucrose gradient into soluble, membrane-associated and pelletable proteins. It had been shown previously for both SecY and LacY that cosedimentation of the in vitro synthesized membrane proteins with INV indeed reflects a functional integration into the membrane (Ahrem et al, 1989;Swidersky et al, 1992). In the absence of INV, only 4% of the SecY synthesized was recovered from the membrane fraction (Figure 4).…”

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase

“…However, it should be noted that antibody to the C-terminal tail of SCY2 (Fig. 11) was very effective for inhibiting the SECA2-independent integration of TIC40 and that E. coli SECY integration does not require preexisting SECY (Swidersky et al, 1992).…”

How proteins cross the bacterial cytoplasmic membrane

“…There is, however, no clear consensus as to whether polytopic membrane proteins use the Sec pathway for membrane integration or not [ 1,64]. Some membrane proteins seem to require SecY and not SecA [146], opening the exciting possibility that SecA can be bypassed and that the SecY/E protein complex can be directly accessed. The SRP and chaperone pathways may thus coexist for general protein export, converge at the SecY/E protein complex and/or differ in the timing of nascent chain association.…”