The diagram at the left summarizes the flow of proteins between the compartments of the endomembrne system. In the initial section of this unit we will consider the transfer of proteins from ER to golgi (red arrow) and the reverse (retrograde) transfer of receptors and proteins destined for retention in the ER compartment (blue arrow). The non highlighted portion of the figure shows the flow of proteins in the secretory, lysosomal and endocytotic pathways that will be considered later. Click on image to enlarge.
To investigate the role of the leader peptide in modulating secretion from living cells, we injected a synthetic peptide into Xenopus oocytes. The peptide consisted of the NH2-terminal leader sequence of mouse immunoglobulin light chain precursor. We found that the leader peptide has two different roles in regulating secretion from the oocytes. First, it competitively inhibits the synthesis of secretory and membrane proteins but not of cytoplasmic proteins. The inhibition occurs both with oocyte proteins and with proteins directed by coinjected myeloma mRNA. The inhibition reaches a maximum 2 hr after injection and decays within 3 hr. It appears to be mediated through the cell membrane, because 125I-labeled leader peptide segregates into the membrane fraction of microinjected oocytes simultaneously with the interference with methionine incorporation. A second role of the microinjected leader peptide is to induce a rapid acceleration in the rate of export of secretory proteins from the oocyte. The maximal enhancement effect is obtained upon injection of 50 ng of leader peptide per oocyte. It is not merely due to the small size, negative charge, or hydrophobicity of the peptide, because enhanced secretion does not occur when glucagon, poly-L-glutamic acid, or Triton X-100 is injected. Furthermore, immunoreaction of the peptide with specific antibodies prior to microinjection prevents the accelerated export. Our observations indicate that in Xenopus oocytes, the leader peptide is involved in both translocation and later step(s) in the secretory pathway.
Proteomic analysis of plasma membrane and secretory vesicles …
AB - The rate of synthesis and the turnover of cytoplasmic membrane proteins were determined in the acinar cells of guinea pig pancreas with the aim of investigating the mechanisms by which the intracellular transport of secretion products occurs. These cells are highly specialized toward protein secretion. By means of in vitro pulse chase experiments and in vivo double labeling experiments, using radioactive L leucine as the tracer, it was found that the turnover of secretory proteins is much faster than that of all membranes involved in their transport (rough and smooth microsome and zymogen granule membranes). Sodium dodecyl sulfate polyacrylamide disk gel electrophoresis of membrane proteins revealed that in each of these membranes there is a marked heterogeneity of turnover; generally the high molecular weight polypeptides have a shorter half life than the low molecular weight polypeptides. These data indicate that the membranes participating in the intracellular transport of secretory proteins are not synthesized concomitantly with the latter. Rather, they are probably reutilized in several successive secretory cycles. The possible relevance of these findings to other secretory systems is discussed.
immediately after their synthesis secretory proteins are ..
15. Figure 16-3, Lodish5e: Experimental demonstration of location of secretory proteins just after synthesis. If cells are incubated with radioactive amino acids for a short period of time, say ten minutes, the radioactive polypeptides produced will be close to their sites of synthesis in the rough ER. If these cells are homogenized, the ER membrane system will be fragmented and these membrane fragments will reseal to form vesicles called microsomes. These microsomes, which are artifacts in the sense that they are created during homogenization, maintain the same membrane orientation as the intact ER, with the membrane associated ribosomes on the outside and the lumenal contents of the ER on the inside of the vesicle. If proteases are added, proteins inside the vesicles are protected from proteolysis since the proteases cannot cross the membrane. But if detergents are added, the membranes are solubilized and the sequestered radioactive proteins are exposed to proteases. The detergent step is an important control, since it shows that proteins are not intrinsically resistant to the proteases but rather require an intact membrane vesicle for resistance.