|Budget Amount *help
¥15,800,000 (Direct Cost: ¥15,800,000)
Fiscal Year 1992: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1991: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1990: ¥9,800,000 (Direct Cost: ¥9,800,000)
SecY is an integral membrane protein and believed to be a central component of the protein translocator in Escherichia coli. We have identified dominant negative mutations of secY whose expression interfered with protein export. Such mutations proved to reside in the C-terminal third of SecY, mostly in the cytoplasmic domain 5 (C5). On of them (secY^d1), a three residue deletion in the C5-TM9 interface, was strongly dominant-negative. The dominant negative mutant protein will sequester the interacting components in an inactive complex, and hence compete with SecY^+ for the formtion of the functiona SecY complex. Linker insertion mutations that suppressed secY^d1 were localized within the C4-TM7 region is important for SecY's interaction with other components of the system.
Consistent with our model, the secY24(Ts) mutation in the C4 domain was found to impair the SecY's interaction with SecE. Also, overproduction of either SecE or "ORF12" in the secDF operon overcame the export interference. Futhermore, we were able to identify a new gene, ydr, as another dosage-dependent overcomer of secY^d1. Although overexpressed (and presumably uncomplexed) SecY was rapidly degraded in vivo, it was significantly stabilized by simultaneous overproduction of either SecE, Orf12,or Ydr. Interestingly, overproduction of Ydr in the secY24 mutant cell severely interfered with protein export and the viability. Ydr, a hydrophilic 181-residue protein with is C-terminal region potentially forming an amphiphilic alpha-helix, is loosely associated with the membrane.
Finally we addressed whether any factors are required for membrane protein integration. Our mutational analyses suggested that FtsH, a putative membrane bound ATPase with homology to a family of eukaryotic ATPases, has a role in assuring efficient anchoring of a hydrophobic stretch to the membrane as well as in the translocation process itself.