Abstract
We have examined the effects of thermosensitive mutations in secA and secY (prlA) genes on the export of proteins to the three layers of the Escherichia coli cell surface. After several hours at the nonpermissive temperature, the export of two major outer membrane proteins, lipoprotein and OmpA, is delayed, then essentially blocked, in either a secA or secY strain. These mutations also have a strong effect on the export of several proteins, such as maltose binding protein, to the periplasm, though the export of many periplasmic proteins is not affected. secA and secY block the assembly of leader peptidase, which is made without a leader sequence, into the inner membrane. However, the membrane assembly of M13 coat protein (an inner membrane protein made with an amino-terminal leader sequence) is not affected. Thus, the requirement for sec function for export does not correlate with the presence or absence of leader peptide or with a particular subcellular compartment, but rather is specific to each particular protein.
Highlights
We have examined the effects of thermosensitive M13 coatprotein (Wickner, 1983), p-lactamase (Koshland mutations in secA and secY genes on the export and Botstein, 1980), and arabinose binding protein
After several hours at the nonpermissive temperature, the export of two major outer membrane proteins, lipoproteinand OmpA,is delayed, essentially blocked, in either a secA or secY strain. These mutations have a strong effect on the export of several proteins, such as maltose binding protein, to the periplasm, though the export of many periplasmic proteins is not affected. secA and secY block the assembly of leader peptidase, which is made without a leader sequence, into the inner membrane
The membrane assembly of M13 coat protein is not affected
Summary
Sec Genes Affect Protein Export-As previously reported (Oliver andBeckwith, 1981), thermalinactivation of secA dramatically slows the exportof pre-maltose binding protein (pre-MBP) and its maturation to MBP (Fig. lA).Cells with a temperature-sensitive mutation insecA were grown a t 30 "C in minimal medium, shifted to 42 "C,and pulse-labeled for 2 min at theindicatedtimesaftertheshift. Pro-OmpA synthesis continued for a t least 2 h, but, as reported (Shiba et al, 1984), with steadily slower kinetics of processing to mature OmpA (Fig. 2B) Eachof these genes affects the export of specific proteins to the periplasm and outer membrane. Fluorographs of SDS-polyacrylamide gel profiles of total cell protein from wild type or secA strains pulse-labeled a t 30 or 42 "C showfew differences; secA is clearly required for lipoprotein maturation. Periplasm prepared from these cellsshows that several proteinsdo require secA for export,butothersdonot (Fig. 3).
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