Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes.

Abstract

The ability of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer into the intravesicular space was investigated by using trypsin as a proteolytic probe. We found that under defined conditions trypsin is able to dissect the translocation activity of microsomal membranes into components that can be separated into two fractions, one soluble and the other membrane bound. The trypsinized membrane fraction has lost its translocation activity. Addition of the trypsin-generated soluble fraction, however, results in reconstitution of translocation activity. These results are compatible with the notion proposed in the signal hypothesis that the translocation activity of the microsomal membrane resides in transmembrane protein(s). We propose that trypsin effects solubilization from the membrane of cytosol-exposed domain(s) involved in recognition of the signal sequence or ribosome or both, leaving behind membrane-integrated domain(s) that provide the environment for the passage of the nascent chain across the membrane. Signal peptidase activity was unaffected by trypsinization of microsomal vesicles consistent with a localization of the active site of this enzyme on the cisternal side of the vesicles.Most of the secretory proteins (1) and several membrane proteins (2-4) so far investigated are synthesized as preproteins containing an amino-terminal extension (signal peptide) when their mRNAs are translated in a cell-free system in the absence of microsomal membranes. The signal peptide portion of the nascent chain has been proposed (5) to contain the necessary information to direct the translating ribosome to form a functional complex with receptor(s) of the rough endoplasmic reticulum membrane and thereby to provide the conditions for a unidirectional translocation of the nascent peptide chain across the membrane into the intracisternal space. Before the nascent chain is completed, the signal peptide is removed by signal peptidase, an endoprotease located on the cisternal side of the membrane (6), except for chicken ovalbumin (7), which is proposed to contain an uncleavable signal sequence (8).Little is known about the recognition-transport machinery in the membrane or the ribosomal components interacting with it. Two membrane proteins, restricted in their location to rough endoplasmic reticulum, have been proposed (9) to function as ribosome receptors, primarily on the basis of their physical association with membrane-bound ribosomes. Furthermore, it has been reported (10) that microsomal membranes can be largely depleted of their translocation activity when extracted with a solution of relatively high ionic strength (500 mM KCl) and that activity can be regenerated when the salt extract is added back to the depleted membranes.We present a way to dissect the translocation activity in the microsomal membrane by limited trypsinization into functionally competent components which were separated by centrifugation. Translocation activity of the membrane can be regenerated by...

The N-terminal region of the 37-kDa translocated fragment of Pseudomonas exotoxin A aborts translocation by promoting its own export after microsomal membrane insertion.

“…Signal peptidase is an integral ER membrane protein whose active site is localized to the lumenal face of microsomes (19,(26)(27)(28)(29). The fact that ssPE280-613 undergoes signal peptide cleavage indicates that at least the N-terminal region of the processed form of the nascent polypeptides is inserted into the membrane to allow the signal sequence cleavage site access to the lumen of microsomes.…”

Mechanisms for the incorporation of proteins in membranes and organelles.

“…In particular, polypeptides that can be released by high salt from normal microsomes (238) (probably after they have been cleaved by endogenous proteasass during preparation of the microsomes) or that can be removed after mild digestion with exogenous enzymes have been shown to be able to restore the translocation capacity of trypsinized or depleted microsomes (237). The active polypeptide released by protease digestion was found to contain a sulfhydryl group that is necessary for restoring the translocation function (102,136,137).…”

“…Nascent polypeptides must be longer than 30-40 amino acids, the length of the segments included within the large ribosomal subunits (18,131) for their signals to emerge from the ribosomes and initiate insertion into the membrane. If, as suggested by its resistance to exogenous proteases, the peptidase that removes signal segments is located on the luminal side of the ER membrane (100,237), the nascent polypeptide would have to attain a minimal length of70-90 amino acids (30-40 plus 20 residues required to cross the membrane, in addition to the 18-30 residues of the signal segment) for the cleavage point to reach the enzyme and the signal segments to be removed . This estimate is substantiated by the steady-state distribution of intact and proteolytically processed nascent chains of secretory polypeptides within bound polysomes (16,156).…”

Cell-free Coupling of Newcastle Disease Virus RNA Transcription, Translation and Co-translational Processing

“…Translation was carried out in a cell-free system obtained from rabbit reticulocyte lysates exactly as described earlier except that the reactions were supplemented with 0.8 mM-fructose 1,6-diphosphate (Content et al, 1978a). Dog pancreas microsomes were prepared essentially as described by Walter et al (1979) with the following modifications: all solutions were supplemented with 50 mM-KC1 and 20 U/ml Trasylol. Rough microsomes were resuspended in 250 mM-sucrose, 50 mMtriethanolamine-HC1 pH 7.5, 1 mM-dithiothreitol, 50 mM-KC1 and i0 U/ml Trasylol (solution A) and incubated with 75 U/ml micrococcal nuclease (Boehringer Corp), 1 mMCaC12 for 15 min at 20 °C.…”