Figure 1a shows the response of growing pigs given diets in which the amount of protein, with a constant amino acid profile, was varied while maintaining a constant energy supply by replacing starch with protein. In addition, the diets were given at three levels of feeding which increased both the protein and energy supply in a fixed ratio. Increasing protein from low and limiting levels at constant energy increased protein deposition in the carcase until energy limited the response. Giving more feed increased the energy supply and allowed the response to dietary protein to continue until the new energy level again became limiting. This will repeat until the genetic potential of the animal or some other factor limits further protein accretion.
Figure 1a. Relationship between increasing protein intake of constant amino acid composition and protein deposition in the carcass of pigs between 20 and 45 kg live weight. The same feeds were fed at three fixed levels of feeding, low, moderate and high. Source: Campbell ., 1985
Radioactive proteins synthesized in an mRNA-dependent reticulocyte cell-free system under the direction of mRNA from AtT-20/D-16v mouse cells were isolated by specific immunoprecipitation using antiserum to either alpha(1-24) corticotropin or beta-endorphin [beta(61-91) lipotropin]. Each immunoprecipitate was fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and shown to contain only one labeled protein with an apparent molecular weight of 28,500. Tryptic peptide analysis of the Mr 28,500 corticotropin and beta-lipotropin molecules isolated from the gels demonstrated that the two proteins had the same lysine, methionine, and tryptophan peptides. Four tryptic peptides from the cell-free product exhibited the same electrophoretic and chromatographic mobilities as marker tryptic peptides from bovine beta-melanotropin and porcine beta-endorphin. The identification of these peptides was confirmed by amino acid composition studies with a variety of labeled amino acids. The beta-lipotropin tryptic peptides were also shown to be located carboxy terminal to the corticotropin tryptic peptides.
Start by keeping in mind that proteins contained in the cisternae of the ER are separated from the cytosol by a membrane. If these proteins are to be moved, they must be moved as part of membrane vesicles, and any enzymes that act on the proteins must be contained in the vesicles or cisternae that contain the proteins.
Proteins are carried from the ER to the Golgi by vesicles (transitional vesicles). These vesicles bud from the ER cisternae through the formation of coated buds as described in the last lecture. There are two major families of coat proteins, COP and clathrin. COP proteins are involved in vesicle formation in the ER and in the cis portion of the Golgi. Clathrin, on the other hand is involved in forming vesicles in the trans Golgi network and at the cell surface.
Bacteria may push protein across the translocation channel, rather than pull it, because the source of energy, ATP, is exclusively cytoplasmic. Once in the periplasmic space, secreted proteins form disulfide bonds and fold into proteinase-resistant conformations. Such folding is necessary before the proteins are transported through holes, poorly described, in the outer membrane.
Figure 1. The SecA system of protein transport. Chaperones bind to a nascent protein in order to keep it an unfolded stal it to receptor SecA at the membrane. SecA translocates the protein across the membrane through the protein pore (SecYl conformational changes.
Some proteins destined for export are recognized in the cytoplasm of E. coli by an entirely different class of signal sequences. In these cases, the sequence is at the C-terminus and is not cleaved off by a signal peptidase after transport. Proteins with this type of signal sequence are closely related to each other and include such proteins as toxins, proteinases, and lipases. The signal sequence is recognized by a different class of plasma membrane protein, ATP-driven protein translocators of the ATP-binding cassette (ABC) family (3, 7). ABC proteins have two cytoplasmic ATP-binding domains and two hydrophobic domains, with six transmembrane sequences. They can be either a single polypeptide or be made up of several polypeptides. A complex of a bacterial ABC export with two accessory proteins allows it to export a cytoplasmic protein across both the inner and outer bacterial membrane at the same time. Assembly of the transporting complex is triggered by substrate binding (8).
For example, it synthesises gylcosaminoglycans (GAGs) which are
chains of a repeating disaccharide (in this case each disaccharide is a six-carbon hexose sugar or hexuronic
acid and a hexosamine) which are added to proteins to make proteoglycans which are exported in vesicles and
secreted to form the extracellular matrix (ECM).
You may be wondering how do the proteins move from the
Unlike bacteria, eukaryotic cells are packed with intracellular membranes. A considerable fraction of those membranes is involved in protein export. Thus protein export in eukaryotes involves translocation across membranes but also transport of the newly synthesized protein through intracellular compartments (9). Protein export in eukaryotes is thus a much more complex process than in bacteria. It becomes necessary to know the intracellular pathway taken by a newly synthesized protein before it reaches the eukaryotic cell surface (see also Protein targeting).
For the most part, eukaryotes do not secrete proteins directly across the plasma membrane. Instead, the newly synthesized proteins are translocated from the cytoplasm into an intracellular organelle, the endoplasmic reticulum (ER). The ER is a series of membranous cisternae and tubules that spread throughout the cytoplasm and is continuous with nuclear membrane. As we shall see, the mechanisms for translocating proteins across ER membranes and bacterial membranes both involve a transmembrane pore. Why have an ER? A common speculation on the evolutionary advantage of the ER is that it provides a controlled milieu in which exported proteins can fold and oligomerize, without being exposed to the rigors of the extracellular world. In this regard, the lumen of the ER resembles the periplasmic space in gram-negative bacteria.
The actual translocation event is through a transmembrane pore. In E. coli the pore is made up of the sec YEG complex, consisting of three major membrane proteins (secY, secE, and secG) with several accessory proteins. The sec YEG complex forms the pore through which unfolded proteins pass on their way out of the cell. Translocation involves pushing the unfolded peptide through the pore by a mechanism that resembles a sewing machine needle or a piston (6). A peripheral membrane protein (secA) takes a 25-residue loop, formed by the amino-terminal signal peptide and the contiguous amino acids, and pushes the loop through the membrane. The amino-terminal amino acids remain cytoplasmic. To do this, the secA protein goes through a remarkable, ATP-driven, conformational change, which causes an arm of the secA protein to be inserted transiently into and partially across the membrane. After the pushing step, ATP is hydrolyzed and secA is returned to a cytoplasmic location, ready for a second translocation pumping cycle. The signal peptide is cleaved off by a "signal peptidase" located in the periplasmic space. Further translocation in 25-residue steps can be driven by additional cycles of protein activity. After the initial step, however, further translocation is facilitated by a transmembrane proton motive force (Fig. 1).