We have reproduced the posttranslational mode of protein translocation across the endoplasmic reticulum membrane with reconstituted proteoliposomes containing a purified complex of seven yeast proteins. This Sec complex includes a heterotrimeric Sec61p complex, homologous to that in mammals, as well as all other membrane proteins found in genetic screens for translocation components. Efficient posttranslational translocation also requires the addition of lumenal Kar2p (BiP) and ATP. The trimeric Sec61p complex also exists as a separate entity that, in contrast with the large Sec complex, is associated with membrane-bound ribosomes. We therefore hypothesize that distinct membrane protein complexes function in co- and posttranslational translocation pathways.

Regulated delivery and removal of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) from postsynaptic elements has been proposed as a mechanism for regulating synaptic strength. Here we test the role of ubiquitin in regulating synapses that contain a C. elegans GluR, GLR-1. GLR-1 receptors were ubiquitinated in vivo. Mutations that decreased ubiquitination of GLR-1 increased the abundance of GLR-1 at synapses and altered locomotion behavior in a manner that is consistent with increased synaptic strength. By contrast, overexpression of ubiquitin decreased the abundance of GLR-1 at synapses and decreased the density of GLR-1-containing synapses, and these effects were prevented by mutations in the unc-11 gene, which encodes a clathrin adaptin protein (AP180). These results suggest that ubiquitination of GLR-1 receptors regulates synaptic strength and the formation or stability of GLR-1-containing synapses.

Endostatin, an inhibitor of angiogenesis and tumor growth, was identified originally in conditioned media of murine hemangioendothelioma (EOMA) cells. N-terminal amino acid sequencing demonstrated that it corresponds to a fragment of basement membrane collagen XVIII. Here we report that cathepsin L is secreted by EOMA cells and is responsible for the generation of endostatin with the predicted N-terminus, while metalloproteases produce larger fragments in a parallel processing pathway. Efficient endostatin generation requires a moderately acidic pH similar to the pericellular milieu of tumors. The secretion of cathepsin L by a tumor cell line of endothelial origin suggests that this cathepsin may play a role in angiogenesis. We propose that cleavage within collagen XVIII's protease-sensitive region evolved to regulate excessive proteolysis in conditions of induced angiogenesis.

We have established an in vitro system for the formation of the endoplasmic reticulum (ER). Starting from small membrane vesicles prepared from Xenopus laevis eggs, an elaborate network of membrane tubules is formed in the presence of cytosol. In the absence of cytosol, the vesicles only fuse to form large spheres. Network formation requires a ubiquitous cytosolic protein and nucleoside triphosphates, is sensitive to N-ethylmaleimide and high cytosolic Ca2+ concentrations, and proceeds via an intermediate stage in which vesicles appear to be clustered. Microtubules are not required for membrane tubule and network formation. Formation of the ER network shares significant similarities with formation of the nuclear envelope. Our results suggest that the ER network forms in a process in which cytosolic factors modify and regulate a basic reaction of membrane vesicle fusion.