SecG, a subunit of the protein translocon, undergoes a cycle of topology inversion. To further examine the role of this topology inversion, we analyzed the activity of membrane vesicles carrying a SecG-PhoA fusion protein (SecG-PhoA inverted membrane vesicles (IMVs)). In the absence of externally added SecA, SecG-PhoA IMVs were as active in protein translocation as SecG ؉ IMVs per SecA. Consistent with this observation, insertion of membrane-bound SecA into SecG-PhoA IMVs was normally observed. On the other hand, externally added SecA did not affect the activity of SecG-PhoA IMVs, but it caused >10-fold stimulation of the translocation activity of SecG ؉ IMVs, indicating that the topology inversion of SecG, which cannot occur in SecG-PhoA IMVs, is essential for cytosolic SecA-dependent stimulation of protein translocation. SecG-PhoA IMVs generated a 46-kDa fragment of SecA upon trypsin treatment. The accumulation of this membrane-inserted SecA in the SecGPhoA IMVs was responsible for the loss of the soluble SecA-dependent stimulation. Moreover, fixation of the inverted SecG topology was found to be dependent on soluble SecA. The dual functions of SecG in protein translocation will be discussed.Most secretory proteins of Escherichia coli are synthesized with an N-terminally extended signal sequence and then posttranslationally translocated across the cytoplasmic membrane via the SecYEG translocon using the driving force provided by translocation ATPase SecA (see recent reviews in Refs. 1 and 2). SecA plays a central role in protein translocation by interacting with many ligands, including presecretory proteins, membrane lipids, SecYEG, and SecDF, and it undergoes a dynamic structure change coupled with protein translocation. SecA, which can be purified as a soluble protein, is inserted deep into the membrane upon the binding of both ATP and a precursor protein, which causes protein translocation of a segment of 20 -30 amino acids (3-5). Thus the translocation of the precursor is thought to be driven by the repeat of the membrane insertiondeinsertion cycle of SecA and to proceed in a stepwise manner. Based on the crystal structures of the translocon and SecA (see Refs. 1 and 2, and references therein), detailed mechanisms of protein translocation are proposed; however, they are derived from a limited number of snapshots of the catalytic cycle. Therefore, many issues, including the oligomeric state of each subunit, structure changes upon protein translocation, mode of action of the translocon, and so on, remain to be clarified.The SecG subunit of the SecYEG translocon possesses two transmembrane stretches with N and C termini exposed to the periplasm (6, 7). We have reported that SecG undergoes a cycle of topology inversion, which couples the SecA cycle with protein translocation (7-10). These phenomena were demonstrated by the translocation-dependent changes in proteinase K (PK) 2 sensitivity of the C-terminal region of SecG and by the chemical labeling of the cysteine-containing SecG mutants, using in vitro and i...

A multicopy suppressor of the cold-sensitive secG null mutation was isolated. The suppressor contained sfa and yccL, the former of which has been reported to be a multicopy suppressor of the fabA6 mutation carried by a temperature-sensitive unsaturated fatty acid auxotroph. Subcloning of the suppressor gene revealed that yccL, renamed gnsA (secG null mutant suppressor), was responsible for the suppression of both the secG null mutation and the fabA6 mutation. In contrast, the sfa gene did not suppress the fabA6 mutation. The ydfY (gnsB) gene, encoding a protein which is highly similar to GnsA, also suppressed both the secG null mutation and the fabA6 mutation. Although both gnsA and gnsB are linked to cold shock genes, the levels of GnsA and GnsB did not exhibit a cold shock response. A gnsA-gnsB double null mutant grew normally under all conditions examined; thus, the in vivo functions of gnsA and gnsB remain unresolved. However, overexpression of gnsA and gnsB stimulated proOmpA translocation of the secG null mutant at low temperature and caused a significant increase in the unsaturated fatty acid content of phospholipids. Taken together, these results suggest that an increase in membrane fluidity due to the increase in unsaturated fatty acids compensates for the absence of the SecG function, especially at low temperature.

GnsA overproduction was previously found to suppress both the secG null mutation and the fabA6 mutation in Escherichia coli by increasing the unsaturated fatty acid contents. We report here that it also increased the acidic phospholipid contents at 20°C but not at 37°C. GnsA overproduction at 20°C specifically inhibited phosphatidylethanolamine synthesis and therefore caused the increase in the proportion of acidic phospholipids.

The Northern Goshawk (Accipiter gentilis Linnaeus 1758), a rare raptor, is listed as an near threatened (NT) species on the red lists by the Ministry of Environment of Japan. Many guidelines about how to manage the forest have been suggested for habitat conservation of the Northern Goshawk, and suggested that active forest management is required to maintain appropriate habitat conditions for extended periods. However, there are few cases of actual active management in Japan. The aim of this study is to evaluate the effectiveness of the active forest management implemented in "The Forest of Accipiter gentilis, Daisen, Tottori Prefecture" by comparing the forest structure and avifauna before and after forest management and to suggest an appropriate management plan. A management plan (Management of pine tree density, improvement by cutting of broad-leaved trees that reached the sub-tall tree layer) was suggested based on the first survey by the Tottori Chapter of the Wild Bird Society of Japan and Tottori University in 2003, and this suggestion was implemented by Tottori Prefecture for the five ensuing years. A second survey, conducted after the management, revealed that pine trees with a large diameter at breast height (DBH) and open spaces between the canopy and understory foliage for flying were preferred as nest areas by the Northern Goshawk. In addition, there was a good foraging range around the case study area. The mosaic of farmland and forest of the Satoyama range was utilized by the Northern Goshawk for foraging. Forest management that includes (1) promoting growth of large diameter trees for nesting and (2) securing flight space in the forest was made clear appropriate for both the conservation of Northern Goshawk habitat and timber cultivation.