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We turn our attention now to the very large class of proteins that are synthesized and sorted in the secretory pathway (see Figure 17-1). Once the ribosomes synthesizing these proteins become bound to the rough ER, the proteins enter or cross the ER membrane cotranslationally — that is, during their synthesis. Soluble proteins in this class first are localized in the ER lumen and subsequently are sorted to the lumen of other organelles or are secreted from the cell. Likewise, the integral membrane proteins in this class initially are inserted into the rough ER membrane during their synthesis; some remain there, but many eventually become localized to the plasma membrane or membranes of the smooth ER, Golgi complex, lysosomes, or endosomes.
Cells are incubated with radiolabeled amino acids and then are homogenized, which fractures the plasma membrane and shears the rough ER into small vesicles (microsomes). The microsomes are purified and treated with a protease in the presence and absence of a detergent. The newly synthesized labeled secretory proteins associated with the microsomes are digested by added proteases only if the permeability barrier of the microsomal membrane is destroyed by detergent. Thus, the newly made proteins are inside the microsomes, equivalent to the lumen of the rough ER.
). When cells are homogenized, the
rough ER breaks up into small closed vesicles, termed rough microsomes, with
the same orientation (ribosomes on the outside) as that found in the intact cell. The simple
experiment outlined in Figure 17-12 shows that
immediately after their synthesis secretory proteins are localized in the lumen of ER vesicles,
although they have been synthesized on ribosomes bound to the cytosolic face of the ER
membrane.
As already noted, all of the proteins that enter the secretory pathway contain an ER signal sequence, generally at the N-terminus (see Table 17-1). This sequence directs the ribosomes that are synthesizing these proteins to the rough ER. Membrane-bound ribosomes and ribosomes free in the cytosol can be separated from other cellular constituents and from each other by a combination of differential and sucrose density-gradient centrifugation (see Figures 5-23 and 5-24). Due to the low buoyant density of phospholipids, membrane-bound ribosomes “band” at a lighter density than do free ribosomes. Biochemical analyses of purified membrane-bound and free ribosomes show that they contain exactly the same proteins and ribosomal RNAs and are functionally indistinguishable. These findings are consistent with the notion that all information for intracellular protein distribution is located in the amino acid sequence of the newly synthesized protein itself.
| Protein Type | Example | Site of Synthesis | ||
|---|---|---|---|---|
| Constitutive Secretory Proteins | ||||
| Serum proteins | Albumin | Liver (hepatocyte) | ||
| Transferrin (Fe transporter) | Liver | |||
| Lipoproteins | Liver, intestine | |||
| Immunoglobulins | Lymphocytes | |||
| Extracellular matrix proteins | Collagen | Fibroblasts, others | ||
| Fibronectin | Fibroblasts, liver | |||
| Proteoglycans | Fibroblasts, others | |||
| Regulated Secretory Proteins | ||||
| Peptide hormones | Insulin | Pancreatic β-islet cells | ||
| Glucagon | Pancreatic α-islet cells | |||
| Endorphins | Neurosecretory cells | |||
| Enkephalins | Neurosecretory cells | |||
| ACTH | Anterior pituitary lobe | |||
| Digestive enzymes | Trypsin | Pancreatic acini | ||
| Chymotrypsin | Pancreatic acini | |||
| Amylase | Pancreatic acini, salivary glands | |||
| Ribonuclease | Pancreatic acini | |||
| Deoxyribonuclease | Pancreatic acini | |||
| Milk proteins | Casein | Mammary gland | ||
| Lactalbumin | Mammary gland | |||
Ribosomes synthesizing proteins bearing an ER signal sequence become bound to the rough ER. As translation is completed on the ER, the polypeptide chains are inserted into the ER membrane or cross it into the lumen. Some proteins (e.g., rough ER enzymes or structural proteins) remain resident in the ER. The remainder move into transport vesicles that fuse together to form new cis-Golgi vesicles. Each cis-Golgi cisterna, with its protein content, physically moves from the cis to the trans face of the Golgi stack (red arrows). As this cisternal progression occurs, many luminal and membrane proteins undergo modifications, primarily to attached oligosaccharide chains. Some proteins remain in the trans-Golgi cisternae, while others move via small vesicles to the cell surface or to lysosomes. In certain cell types (e.g., nerve cells and pancreatic acinar cells), some soluble proteins are stored in secretory vesicles and are released only after the cell receives an appropriate neural or hormonal signal (regulated secretion). In all cells, certain proteins move to the cell surface in transport vesicles and are secreted continuously (constitutive secretion). Like soluble proteins, integral membrane proteins move via transport vesicles from the rough ER to the cis-Golgi and then on to their final destinations. The orientation of a membrane protein, established when it is inserted into the ER membrane, is retained during all the sorting steps: Some segments always face the cytosol; others always face the exoplasmic space (i.e., the lumen of the ER, Golgi cisternae, and vesicles or the cell exterior). Retrograde movement via small transport vesicles retrieves ER proteins that migrate to the cis-Golgi and returns them to the ER. Similarly, cis- or medial-Golgi proteins that migrate to a later compartment are retrieved by small retrograde transport vesicles. [See B. Glick and V. Malhotra, 1988, Cell 95:883.]
outlines the movement of proteins within
the secretory pathway. Most newly made proteins in the ER lumen or membrane are incorporated
into small, ≈50-nm-diameter transport
vesicles. These either fuse with the cis-Golgi or with each other to
form the membrane stacks known as the cis-Golgi reticulum (network). From the
cis-Golgi certain proteins, mainly ER-localized proteins, are retrieved to
the ER via a different set of retrograde transport vesicles. In the process
called cisternal migration, or cisternal progression, a new
cis-Golgi stack with its cargo of luminal protein physically moves from the
cis position (nearest the ER) to the trans position
(farthest from the ER), successively becoming first a medial-Golgi cisterna
and then a trans-Golgi cisterna. As this happens, membrane and luminal
proteins are constantly being retrieved from later to earlier Golgi cisternae by small
retrograde transport vesicles. By this process enzymes and other Golgi resident proteins come
to be localized either in the cis- or medial- or
trans-Golgi cisternae.
These mutants can be grouped into five classes, corresponding to the site where newly made secretory proteins (red dots) accumulate when cells are shifted from the growing (permissive) temperature to the higher nonpermissive one. Analysis of double mutants permitted the sequential order of the steps to be determined. [See P. Novick et al., 1981, Cell 25:461; C. A. Kaiser and R. Schekman, 1990, Cell 61:723; and N. Green et al., 1992. J. Cell Biol. 116:597.]
).
To determine the order of the steps in the pathway, researchers analyzed double sec mutants. For instance, when yeast cells contain mutations in both class B and class D functions, proteins accumulate in the rough ER, not in the Golgi cisternae. Since proteins accumulate at the earliest blocked step, this finding shows that class B mutations must act at an earlier point in the maturation pathway than class D mutations do. These studies confirmed that as a secretory protein matures it moves sequentially from the cytosol → rough ER → ER-to-Golgi transport vesicles → Golgi cisternae → secretory vesicles and finally is exocytosed.
As noted above, a newly formed cis-Golgi vesicle, with its luminal protein cargo, progresses from the cis face to the trans face of the Golgi complex and then into the trans-Golgi reticulum. At one time it was thought that secreted proteins move from the cis- to the medial-Golgi, and from the medial- to the trans-Golgi, via small transport vesicles. Indeed there are many small vesicles that move proteins from one Golgi compartment to another, but they appear to do so in the reverse, or retrograde, direction; these vesicles retrieve ER or Golgi enzymes to an earlier compartment in the secretory pathway. In this way enzymes that modify secretory proteins come to be localized in the correct organelle.
The first evidence for the cisternal progression model of Golgi function came from careful microscopic analysis of the synthesis of algal scales. These are cell-wall glyco-proteins that are assembled in the cis-Golgi into large complexes visible in the electron microscope. Like other secretory proteins, newly-made scales move from the cis- to the trans-Golgi, but they can be 20 times the size of the ≈50-nm-diameter transport vesicles that bud from Golgi cisternae. Thus it was thought unlikely that these and other secretory proteins move from one Golgi compartment to another via small vesicles.
Similarly, in the synthesis of collagen by fibroblasts, large aggregates of the procollagen precursor often form in the lumen of the cis-Golgi. These aggregates are too large to be incorporated into small transport vesicles, and investigators could never find such aggregates in transport vesicles. In one test of the cisternal progression model, collagen folding was blocked by an inhibitor of proline hydroxylation, and soon all pre-made, folded, procollagen aggregates were secreted from the cell. When the inhibitor was removed, newly made procollagen peptides folded and then formed aggregates in the cis-Golgi that subsequently could be seen to move as a “wave” from the cis- through the medial-Golgi cisternae to the trans-Golgi, followed by secretion and incorporation into the extracellular matrix. Procollagen aggregates were never seen in small transport vesicles. Together with other evidence, decribed later, that the small transport vesicles near the Golgi are moving proteins in the retrograde direction, most researchers in the field have come to favor the cisternal progression model.
The pathway for the maturation of secretory proteins elucidated by autoradiographic, genetic, and electron microscope studies in yeasts, algae, fibroblasts, and pancreatic acinar cells is thought to function in all eukaryotic cells. As we detail in later sections, each step in the pathway requires the action of multiple proteins.
). These
plasma-membrane glycoproteins also have been shown to undergo the same types of modifications
in the same ER and Golgi compartments that secretory proteins do.Although all cytosolic ribosomes are functionally equivalent, membrane-attached and membraneunattached ribosomes synthesize different classes of proteins, depending on a signal sequence in the protein itself.
Newly made secretory proteins are localized to the lumen of the rough ER.
All mammalian cells continuously secrete certain proteins, such as those in the extracellular matrix.
Certain cell types store proteins such as hormones and digestive enzymes in secretory vesicles, awaiting a neural or hormonal signal that triggers an elevation in cytosolic Ca2+ and then protein secretion.
).Small transport vesicles bud off from the ER and fuse to form the cis-Golgi reticulum. By cisternal migration, cis-Golgi vesicles with their luminal protein cargo move through the Golgi complex to the trans-Golgi reticulum. Proteins are retrieved from the cis-Golgi to the ER and also from later Golgi cisternae to earlier ones by small retrograde transport vesicles.
Plasma-membrane glycoproteins follow the same maturation pathway as continuously secreted proteins.
Both secreted and integral membrane proteins undergo various modifications as they mature in the secretory pathway.
