Many vesicles similar in size to rough microsomes, but lacking attached ribosomes, are also found in cell homogenates. Such smooth microsomes are derived in part from smooth portions of the ER and in part from vesiculated fragments of the plasma membrane, Golgi apparatus, endosomes, and mitochondria (the ratio depending on the tissue). Thus, whereas rough microsomes are clearly derived from rough portions of ER, it is not easy to separate smooth microsomes derived from different organelles. The smooth microsomes prepared from liver or muscle cells are an exception. Because of the unusually large quantities of smooth ER or sarcoplasmic reticulum, respectively, most of the smooth microsomes in homogenates of these tissues are derived from the smooth ER or sarcoplasmic reticulum. The ribosomes attached to rough microsomes make them more dense than smooth microsomes. As a result, we can use equilibrium centrifugation to separate the rough and smooth microsomes (Figure 12–34B). Microsomes have been invaluable in elucidating the molecular aspects of ER function, as we discuss next.
signal sequences Were first Discovered in proteins imported into the rough er
The ER captures selected proteins from the cytosol as they are being synthesized. These proteins are of two types: transmembrane proteins, which are only partly translocated across the ER membrane and become embedded in it, and water-soluble proteins, which are fully translocated across the ER membrane and are released into the ER lumen. Some of the transmembrane proteins function in the ER, but many are destined to reside in the plasma membrane or the membrane of another organelle. The water-soluble proteins are destined either for secretion or for residence in the lumen of the ER or of another organelle. All of these proteins, regardless of their subsequent fate, are directed to the ER membrane by an ER signal sequence, which initiates their translocation by a common mechanism.
Signal sequences (and the signal sequence strategy of protein sorting) were first discovered in the early 1970s in secreted proteins that are translocated across the ER membrane as a first step toward their eventual discharge from the cell. In the key experiment, the mRNA encoding a secreted protein was translated by ribosomes in vitro. When microsomes were omitted from this cell-free system, the protein synthesized was slightly larger than the normal secreted protein. In the presence of microsomes derived from the rough ER, however, a protein of the correct size was produced. According to the signal hypothesis, the size difference reflects the initial presence of a signal sequence that directs the secreted protein
Figure 12–34 The isolation of purified rough and smooth microsomes from the ER. (a) a thin section electron micrograph of the purified rough er fraction shows an abundance of ribosome-studded vesicles. (B) When sedimented to equilibrium through a gradient of sucrose, the two types of microsomes separate from each other on the basis of their different densities. note that the smooth fraction will also contain non-er-derived material.  (a, courtesy of George palade.)
Figure 12–35 The signal hypothesis.
a simplified view of protein translocation across the er membrane, as originally proposed. When the er signal sequence emerges from the ribosome, it directs the ribosome to a translocator on the er membrane that forms a pore in the membrane through which the polypeptide is translocated. a signal peptidase is closely associated with the translocator and clips off the signal sequence during translation, and the mature protein is released into the lumen of the er immediately after its synthesis is completed. The translocator is closed until the ribosome has bound, so that the permeability barrier of the er membrane is maintained at all times.
to the ER membrane and is then cleaved off by a signal peptidase in the ER membrane before the polypeptide chain has been completed (Figure 12–35). Cell-free systems in which proteins are imported into microsomes have provided powerful procedures for identifying, purifying, and studying the various components of the molecular machinery responsible for the ER import process.