Cytoplasm-free cell envelopes

The chemotaxis protein CheY was subsequently identified by both the whole-cell and reductionist approaches as the clockwise signal, i.e., as the molecule that interacts with the switch, thereby promoting clockwise rotation. In the whole-cell approach, second-site suppression analysis implied that CheY interacts with the switch (for reviews, see [56, 459]), and overproduction of CheY in a gutted strain generated clockwise rotation ([168] for a review, see reference [212]). In the reductionist approach, purified CheY inserted into cytoplasm-free envelopes caused some of the envelopes to rotate clockwise [594]. The absence of cytoplasmic chemotaxis proteins in the intact cells and the absence of cytoplasm in the envelopes indicated that the interaction of CheY with the switch is direct. Later biochemical studies demonstrated... 

Entry of the macromolecule into the cytoplasm is a process of very low efficiency, however it is known to occur with various materials, e.g., viral infection can be achieved by exposing cells to some viral nucleic add in the presence of a basic polymer or at high concentrations of calcium ions in the medium375. In a latter part of this review we will present evidence to the point that foreign macromolecules free in the cytoplasm slowly undergo the autophagy process in which they are converted into an enveloped form. 

Fig. 3 Isolation of nuclei from suspension tissue culture cells. (A) Low-power SEM image of nucleus isolated from K.S62 tissue culture cell, after isolation and Percoll density-gradient purification. This particular nucleus shows surface indentations due to its position in the cell cycle (approaching prophase), but the overall surface morphology of the nuclear envelope is largely free of cytoplasmic contamination, and numerous pore complexes (arrowed) arc apparent. Scale bar, 1.0 /u,m. (B) Detail of A, showing cytoplasmic surface of the nuclear envelope with clearly visible nuclear pore complexes, and a typical density of ribosomes (remember the outer nuclear envelope is the innermost element of the rough endoplasmic reticulum). Scale bar, 125 nm.

Lohka. M. J., and Masui, Y. (1984). Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell free preparations from amphibian eggs. J. Cell Biol. 9si 1222-1230. 

The 30 kDa protein is encoded by nuclear DNA, and precursor protein is transported from cytoplasm to chloroplast. As the protein is located at the lumenal surface of the thylakoid, it must also cross thylakid membrane before it is incorporated into the PS II complex properly. Identification of the precursor protein has been carried out with isolation of mRNA and identification of the translation product. Transit peptide of several kilodalton is shown to be attached to the amino terminal of the mature protein. We tried to identify the precursor protein of the 30 kDa protein with Euglena cells whose chloroplast is functionally inactivated. Treatment of the cells with streptomycin under illumination induced a protein of 36 and 52 kDa which were cross-reactable with the antibody against spinach 33 kDa protein. The proteins were also detected when chloroplast-less mutant was illuminated. The 36 kDa protein is probably a precursor of the 30 kDa protein. The 52 kDa protein was dissociated into 30 kDa protein and some other protein(s) with SDS-PAGE with 8 M urea, and it is possible that we detect a complex form of the precuresor or mature 30 kDa porotein and the protein(s) which may stabilize the free 30 kda protein or assist the 30 kDa protein in transportation across the envelope and/or thylakoid membranes. Analysis is now being carried out to elucidate the characteristics of the precursor proteins. 

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