Faculty Bibliography

Dog pancreas rough microsomes were solubilized in 1% octyl beta-glucoside, and membrane vesicles were reconstituted by slow 30-fold dilution with a buffer of low ionic strength. Asymmetric assembly of the membranes occurred during reconstitution since the vesicles formed contained ribosomes bound only to the vesicular outer surfaces. The reconstituted vesicles were similar in protein composition to native rough microsomes, although these vesicles were largely devoid of luminal-content proteins. These reconstituted vesicles could translocate and process nascent secretory (human placental lactogen) and membrane proteins (influenza hemagglutinin and rat liver ribophorin I) synthesized in cell-free translation systems programmed with the corresponding mRNAs. Signal cleavage and N-glycosylation only occurred when the reconstituted membranes were present during translation, providing evidence that the translocation apparatus was asymmetrically assembled into the reconstituted membranes. When a supernatant lacking ribosomes and particles greater than 50S from centrifuging the detergent-solubilized microsomes at high speed was used for reconstitution, smooth-surfaced membrane vesicles were obtained that, except for the absence of ribosomal proteins, were similar in protein composition to that of the reconstituted vesicles from total solubilized rough microsomes. The reconstituted smooth-surfaced vesicles, however, were totally inactive in cotranslational processing and translocation of nascent polypeptides. These findings suggest that ribosomes and/or large macromolecular complexes, not dissociated under our solubilization conditions, are essential for in vitro assembly of a functional translocation apparatus

When synthesized in polarized epithelial cells, the envelope glycoproteins hemagglutinin of influenza and G of vesicular stomatitis virus are targeted to the apical and basolateral plasma membranes, respectively. To determine which portions of these transmembrane proteins contain information necessary for their sorting, the behavior of two different G-hemagglutinin chimeric polypeptides, consisting of all or nearly all the luminal portion of the vesicular stomatitis virus G protein linked to C-terminal segments of influenza hemagglutinin that included its transmembrane and cytoplasmic domains, was studied in MDCK cells transformed with the corresponding cDNAs. Both chimeras were transported from the endoplasmic reticulum to the Golgi apparatus and from there to the cell surface with the same rapid kinetics as the intact G protein. By using a cell surface immunoprecipitation assay with monolayers cultured on permeable filters that allows the recovery of labeled protein molecules present in each cell surface domain, it was found that both chimeric proteins as well as the intact G protein were delivered almost exclusively to the basolateral surface. This polarized distribution of the polypeptides did not change during a subsequent 90-min chase period, although during this time a large fraction of the glycoprotein molecules underwent degradation. In addition, a small fraction of the cell surface-associated glycoprotein molecules shed their ectoplasmic segments into the basolateral compartment, apparently as a result of a proteolytic cleavage. Immunofluorescence on transverse frozen sections and immunoelectron microscopy revealed a prominent accumulation of the chimeric polypeptides in the lateral cell membranes, with lesser amounts on the basal and apical surfaces. These results indicate that information specifying the basolateral transport of the G glycoprotein is located within the first 426 N-terminal amino acids of its ectoplasmic portion

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation

Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino-terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein

The major characteristic of the eucaryote cell is the presence of specialized organelles in which macromolecular components responsible for various subcellular functions are segregated. The membranes of these organelles serve not only as divisions between the various cytoplasmic compartments, but also provide scaffolding within which the macromolecular complexes of the organelle assemble and become functionally integrated. It is obvious that because of the degree of complexity resulting from the existence of numerous compartments and membrane systems, the development of a genetic programme in a eucaryote cell requires not only the transcription of specific genes and translation in the cytoplasma of the resultant messenger RNA, but also the activity of mechanisms which ensure that each polypeptide reaches the site of its function, which may be in the cytosol, in a membrane, or in the luminal cavity of an organelle. In the special case of membrane proteins, such mechanisms must result not only in the specific distribution of polypeptides newly synthesized in the various types of cell membrane, but also the arrangement of them required in the lipid bi-layer necessary for their normal function