Membrane proteins can pass through the membrane once or many times. In addition some membrane proteins are oriented so that their amino terminus is in the cytosol, and their carboxy terminus is extracellular, while others have the opposite orientation. And some proteins have both ends on the same side of the membrane. (Fig. 17-21 in Lodish et al.) What determines whether a protein is embedded in a membrane and what determines its orientation?
Proteins with an internal hydrophobic "stop transfer sequence" are inserted N-terminus first into the ER membrane, as are secreted proteins. However, when the hydrophobic stop transfer segment is inserted into the translocon--the proteinaceous pore in the ER membrane, the protein somehow gets "stuck" in the pore, so that it no longer moves through the membrane (Fig. 17-22). Although ribosome continues to add amino acids to the protein, these amino acids remain in the cytosol, and eventually the protein is completed and escapes from the translocon, becoming an integral membrane protein, but how this happens is not clear (at least not to me.) Since the lumen of the ER is topologically equivalent to the exoplasmic space (see Fig. 17-13), a protein that has its N terminus in the ER lumen and its C terminus in the cytosol will end up with its N terminus protruding out of the cell and its C terminus still in the cytosol. This is more or less the "default" pattern for membrane proteins. Unless they contain some additional information that enables them to pass through the membrane more than once, or to insert in a different manner, they are single pass proteins with an extracellular N-terminus.
Of course, many membrane proteins have different orientations (Fig. 17-21) because they contain information that orients them in a different way. One group of proteins lacks an N-terminal hydrophobic signal sequence, but contains an internal sequence that serves the same function (Fig. 17-23). In this case the protein is synthesized on a free ribosome until its signal region is made, which is then latched onto by a SRP, and then targeted to the translocon in the usual way. However, when this happens, the internal hydrophobic sequence serves two functions. First it a "signal sequence" that gets the protein to the ER membrane, but it's also a "stop-transfer" sequence, that anchors it in the membrane once the protein arrives there. It appears that the N terminus of these proteins remains in the cytosol, and the C terminus is slipped through the translocon into the lumen of the ER.
Some proteins that contain an internal hydrophobic/membrane anchoring sequence nonetheless end up with their N terminus on the cytosolic side of the membrane, unlike the asialogylcoprotein receptor shown in Fig. 17-23. What apparently determines whether the protein is inserted one way or the other is the precise nature of the hydrophilic amino acids that flank the two ends of the hydrophobic sequence. (There must be hydrophilic amino acids at the ends of the hydrophobic sequence: why?) In general, it's been found that the end of the hydrophobic sequence with the most positively charged amino acids ends up in the cytosol; presumably that is in part determined by the fact that the cytosol is somewhat negatively charged overall compared to the external space or ER lumen.
Multiple pass transmembrane proteins, like the widespread 7-transmembrane G-protein coupled receptor proteins that we'll be hearing much more about later, are made up of alternating hydrophilic and hydrophobic regions. The first hydrophobic sequence binds the SRP and is inserted into the translocon, where it also serves as a stop transfer sequence, just as in the single-pass proteins (Fig. 12-24). As the protein is elongated by the ribosome, it moves through the translocon until the second hydrophobic sequence is made, and this serves as second stop transfer sequence. Now the part of the protein between the first and second stop transfer sequences is trapped on one side of the ER membrane (usually the lumenal side). Subsequent pairs of hydrophobic sequences serve as membrane anchoring and stop transfer sequences, but because they are being originally synthesized in the cytosol, they must somehow insert into the lipid bilayer, presumably with the help of a translocon pore, but without any further assistance from a Signal Recognition Particle.
The orientation of a membrane protein (or at least the number of transmembrane segments) can often be accurately predicted from a hydropathy plot, which is useful in determining which parts of a protein are likely to be associated with the lipid bilayer. This shows what the free energy requirement is to place various parts of the protein, as measured along its amino acid backbone, into water. In such a plot a negative value indicates areas of the protein that have negative free energy in water; those are hydrophilic. Positive values indicate areas of positive free energy that are hydrophobic, and therefore are likely to be membrane-spanning segments of the protein.
Protein Modification in the ER and Golgi apparatus:
Once a proteins are synthesized and inserted into the lumen of the ER or into the ER membrane, they can undergo a series of modifications and rearrangements in the ER-Golgi-secretory vesicle pathway that occurs nowhere else in the cell. These modifications include addition of oligosaccharide side chains, often extensive remodeling of the side chains, proteolytic cleavage of the protein, and sometimes covalent attachment of lipid molecules. In addition, proteins are folded into their proper three dimensional structures in the ER, and those that fail to fold properly are often destroyed, a process called "quality control", by analogy to a manufacturing facility, where defective products are supposed to be pulled from distribution.
The lumen of the ER is what is called a "oxidizing environment", which means that the reduced sulfhydyl groups on the side chains of cysteine are often oxidized to disulfide bonds, thus helping to mold the final three dimensional shape of the protein; formation of disulfide bonds occurs only in the ER, and thus cytosolic proteins don't have disulfide bonds.
As I mentioned earlier, proper folding of proteins in the ER lumen usually involves chaperone proteins like Hsc70, BiP and several others mentioned in the textbook. These proteins are not only involved in initial folding of the protein, but can often assist erroneously folded proteins to rearrange into the proper conformation. In addition individual polypeptide chains are combined with others to form multimeric (i.e., many subunit) proteins in the ER.
If proteins are not properly folded, it is believed that they remain in the ER, and don't pass on to the Golgi apparatus with the other proteins. They remain in the ER lumen, attached to chaperone proteins like Hsc70, which essentially provides repeated opportunities for the protein to interact with the ER proteins that promote catalytic refolding of the improperly folded protein. (If many proteins are improperly folded in the ER, the cell actually produces more of the chaperone and folding proteins to try to overcome the problem. One of the easiest ways to cause protein misfolding is to subject an organism to abnormally high temperatures, around 40oC (104o F, equivalent to a high fever). This "heat shock" induces protein misfolding, which in turn causes synthesis of the chaperone/folding proteins. These chaperone/folding proteins are thus turned on by a "heat shock", which is how many were discovered and named. For example Hsc70 means "heat shock chaperone of 70kDa molecular weight). After an hour or so, if the misfolded protein doesn't get itself straightened out, the cell gives up on it and degrades it. By a process that is not well understood, the protein is pushed out of the ER lumen back into the cytosol, through a translocon, where it is ubiquinated and degraded by the complex of protein-degrading enzymes called a proteosome.
Proteins that fold properly in the ER are then carried by vesicles on to the Golgi apparatus, as we'll see shortly. However, some proteins are needed in the ER itself. It would be a good self test if you could think of some of these "ER resident" proteins before you read on. Some examples of proteins that are permanent residents of the ER include the signal peptidase, Hsc70, glycosylating enzymes (see below), folding enzymes, etc. Yet in the lumen of the ER these are mixed together with proteins destined for secretion. Thus, somewhere in the secretory pathway there must be a place where the proteins that will be secreted are separated from those that will remain in the ER (or in the Golgi apparatus). For ER resident proteins the sorting takes place in the Golgi apparatus. Non only do vesicles pick up material from the ER and carry it to the Golgi, but there are also vesicles that move from the Golgi back to the ER. (A good thing, since otherwise all the ER membrane would get moved over to the Golgi.) These "retrograde" (i.e., backward-moving) vesicles carry some proteins back to the ER. Its pretty clear that what distinguishes the proteins that will go back to the ER from those that move on through the secretory pathway is the presence of a "retention sequence" in the protein, consisting of the four amino acids lysine-aspartate-glutamate-leucine (KDEL in the single letter abbreviation for amino acids). This implies that the Golgi membrane contains a protein that binds KDEL (a "KDEL receptor") that is somehow targeted into the vesicles that will move retrogradely back to the ER. A good PhD thesis would be to figure out what it is about the KDEL receptor that gets it into the appropriate retrograde vesicles, because nobody yet knows.
Most, though not all, proteins that enter the ER acquire covalently attached oligosaccharide; that is, short chains of sugar (carbohydrate) molecules. One common kind of modification is the addition of a group of sugars to asparagine side chains--so-called N-linked oligosaccharides. These consist of a branched chain arrangement of glucose, mannose (another 6 C sugar), and N-acetylglucosamine (GlcNAc) (Figure 17-30 and 17-31). This oligosaccharide is synthesized first by stepwise addition of sugar residues on to a lipid named dolichol phosphate in the membrane of the ER, by a series of enzymes, partly in the cytosol and partly in the ER lumen. There are thus normally a bunch of the glycolipids (oligosaccharide attached to dolichol phosphate) sitting in the ER membrane (Fig. 17-35) . When a protein is synthesized and exposes a glycosylation sequence in the lumen of the ER (which is asn-X-ser or asn-X-thr), then an enzyme, oligosaccharyl transferase, moves the whole 14-sugar oligosaccharide unit from the glycolipid to the glycoprotein. Almost all membrane and secretory proteins are modified in this way as they are made, and usually some of the added sugar moieties are removed by enzymes in the ER before the protein moves on to the Golgi apparatus. (Fig. 17-36).
A second kind of glycosylation involves addition of sugars to the side chains of serine and threonine residues of the protein--and these are called O-linked oligosaccharides. (Rarely proteins contain a modified form of lysine, called hydroxylysine, that can also serve as an oligosaccharide acceptor group). In contrast to N-linked oligosaccharides, which are made and transferred as a unit to the protein, O-linked oligosaccharides are added one at a time directly onto the protein by enzymes that are integral ER membrane proteins (See Figs. 17-32 and 33).
A final kind of interesting modification is the covalent transfer of most of a membrane protein in the ER to a lipid molecule called glycosylphosphatidylinositol (GPI). In these cases the protein, anchored in the membrane by its signal sequence, is cleaved by an enzyme that breaks the peptide chain, then reforms an amide bond with the lipid. The protein remains anchored to the membrane by the lipid, rather than by its own signal sequence, but it is readily released by an enzyme, called phospholipase C, that cuts between the diglycerol phosphate (phosphatide) and the inositol bond.
Lastly, the ER is not only the site of synthesis and modification of membrane and secretory
proteins, but its also the place where most lipids for all the membranes of the cell are made, especially in the smooth ER. The process by which lipids made in the smooth ER are carried to the appropriate cellular organelles is even more mysterious than the mechanisms of protein targeting.
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