Figure 18.3. Scheme illustrating the orientation of the subunits of the membrane-bound 80S ribosome relative to the surface of the endoplasmic reticulum membrane. (P. N. T. Unwin, J. Mol. Biol. 132, 69-84, 1979; A. K. Christensen, Cell Tissue Res. 276, 439-444, 1994).

described above (Section 18.2), or in the hydrophobic environment of the membrane lipid bilayer for membrane-bound polyribosomes.

As was noted years ago, free polyribosomes synthesize primarily water-soluble proteins for housekeeping use of the cytoplasm, whereas the membrane-bound particles synthesize either the proteins for incorporation into membranes or the secretory proteins which are transported out of the cell through the membranes (Siekevitz & Palade, 1960; Redman et al, 1966; Redman, 1969; Ganoza & Williams, 1969; Morrison & Lodish, 1975). The soluble cytoplasmic proteins synthesized on free polyribosomes are folded in the aqueous medium as they emerge from the ribosomes. As a consequence, they form a typically globular structure, with a more or less polar surface and a hydrophobic core. In contrast, protein synthesis on membrane-bound ribosomes causes the growing polypeptide to come into contact with the hydrophobic milieu of the membrane lipid bilayer. In the case of proteins destined to become components of a given membrane (the endoplasmic reticulum membrane of Eukaryotes or the plasma membrane of bacteria), the hydrophobic environment dictates the mode of their folding, with numerous hydrophobic residues being exposed outside the molecule. The trans-membrane hydrophobic sequences of such proteins often exist in the a-helical conformation (consider, for example, the case of bacteriorhodopsin - Henderson & Unwin, 1975; see also Fig. 13.3 for the membrane-bound chloroplast reaction center protein D1).

With proteins transported through the membrane, however, the picture appears to be more complex. The nascent chain passing through the membrane is finally folded in the aqueous milieu of the endoplasmic reticulum lumen in Eukaryotes, or in the periplasmic space of gram-negative bacteria, or in the external medium for other bacteria. The transmembrane translocation of such polypeptides is accompanied by their multistage folding coupled with co-translational processing and covalent modifications.

18.4. Interaction of Translating Ribosomes with Membranes 18.4.1.Early Observations

The idea that protein synthesis on membrane-bound ribosomes is coupled with transmembrane protein translocation emerged from observations on the intimate association of nascent polypeptide chains with membranes of the rough endoplasmic reticulum in eukaryotic cells (Sabatini & Blobel, 1970) and with the plasma membrane in bacteria (Smith et al., 1978a). The translating ribosomes were found to be anchored firmly on the membrane by the growing peptide. Only puromycin treatment, which resulted in the abortion of the peptide from the ribosomes, allowed the complex dissociation into free ribosomes and membranes, leaving the peptide in the membrane. Thus, it became clear that the growing peptide significantly contributes to the association between the translating ribosome and the membrane. A rupture of this anchor by puromycin in the bacteria results in the immediate release of the ribosomes from the membranes.

In contrast, with eukaryotic cells, after the peptide anchor is broken the ribosomes still show a marked affinity to endoplasmic reticulum membranes. The complete dissociation of the ribosomes from endoplasmic reticulum membranes in vitro may be achieved only by combining the treatment of microsomes with puromycin and high ionic strength solutions (Adelman et al., 1973; Harrison et al., 1974). It also can be demonstrated that non-translating ribosomes, or ribosomes that have just started translation and contain only a short peptide have a certain affinity to endoplasmic reticulum membranes. From all this, it was assumed that the membranes of the rough endoplasmic reticulum contain specialized receptors which are responsible for the reversible association of ribosomes with a membrane; these receptors were also thought to help the membrane to accept the ribosome-bound nascent peptides and thus to form membrane "pores" (or intra-membrane tunnels) for the growing polypeptide chains.

By now, numerous experimental facts concerning interactions of translating ribosomes and ribosome-bound nascent polypeptides with membranes and their structural elements have been accumulated, and well-substantiated models for ribosome-membrane and nascent-peptide-membrane recognition, as well as co-translational trans-membrane translocation of nascent polypeptide have been proposed (reviewed in Harwood, 1980; Inouye & Halegoua, 1980; von Heijne, 1988; High & Stirling, 1993; Walter & Johnson, 1994; Rapoport et al., 1996; Martoglio & Dobberstein, 1996; Corsi & Schekman, 1996; Johnson, 1997).

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