Involvement of SecDF and YidC in the membrane insertion of M13 procoat mutants.
The M13 phage Procoat protein is one of the best characterized substrates for the novel YidC pathway. It inserts into the membrane independent of the SecYEG complex but requires the 60 kDa YidC protein. Mutant Procoat proteins with alterations in the periplasmic region had been found to require SecYEG and YidC. In this report, we show that the membrane insertion of these mutants also strongly depends on SecDF that bridges SecYEG to YidC. In a cold-sensitive mutant of YidC, the Sec-dependent function of YidC is strongly impaired. We find that specifically the SecDF-dependent mutants are inhibited in the cold-sensitive YidC strain. Finally, we find that subtle changes in the periplasmic loop such as the number and location of negatively charged residues and the length of the periplasmic loop can make the Procoat strictly Sec-dependent. In addition, we successfully converted Sec-independent Pf3 coat into a Sec-dependent protein by changing the location of a negatively charged residue in the periplasmic tail. Protease mapping of Pf3 coat shows that the insertion-arrested proteins that accumulate in the YidC- or in the SecDF-deficient strains are not translocated. Taken together, the data suggest that the Sec-dependent mutants insert at the interface of YidC and the translocon with SecDF assisting in the translocation step in vivo.
Chen
Minyong
M
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Xie
Kun
K
Yuan
Jijun
J
Yi
Liang
L
Facey
Sandra J
SJ
Pradel
Nathalie
N
Wu
Long-Fei
LF
Kuhn
Andreas
A
Dalbey
Ross E
RE
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics* / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism / Q000382:microbiology / Q000821:virology; D003080:Cold Temperature; D029968:Escherichia coli Proteins / Q000502:physiology*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D019897:Periplasm / Q000378:metabolism / Q000821:virology; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics
D036022:Capsid Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C499394:SecD protein, E coli; C489583:SecF protein, E coli; C412443:YIDC protein, E coli; C025668:coat protein, Bacteriophage M13