Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

Dynein is a minus end-directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid-1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The recently described retrograde transport route is a highly selective pathway that allows some internalized molecules to reach the trans‐Golgi network from early/recycling endosomes, bypassing the recycling route to the plasma membrane and the late endocytic pathway. The non‐toxic receptor‐binding B‐subunit of bacterial Shiga toxin has played an important role in the discovery and molecular dissection of membrane trafficking at the early/recycling endosomes–TGN interface. This unit describes several recent methods for quantitative biochemical and morphological analysis of retrograde transport. The sulfation assay permits the detection and quantification of cargo protein transport from endosomes to the TGN, describing how sulfation‐site peptides can be chemically coupled to cargo proteins. Furthermore, a variant of the sulfation assay on permeabilized cells is presented. The chemical crosslinking theme is extended to horseradish peroxidase for the ultrastructural study of the Shiga toxin–containing early/recycling endosomes by whole mount analysis. Finally, an endocytosis assay describes concomitant analysis of cellular uptake of Shiga toxin and transferrin.

One of the first steps in the infective cycle of an enveloped virus consists of the fusion of the viral and cellular membranes. This process is usually achieved as a result of membrane destabilization brought about by a viral fusion peptide located at the amino terminus of one of the viral envelope glycoproteins. Previous sequence similarity studies by Rodrlguez-Crespo et al. (Journal of General Virology 75, 637-639, 1994) have shown that a hydrophobic stretch in the amino-terminal sequence of the S protein of hepatitis B virus shares several characteristics with fusion peptides of retroviruses and paramyxoviruses. A 16 residue peptide with this sequence was synthesized and its interaction with liposomes characterized. This peptide was able to mediate vesicle aggregation, lipid mixing and liposome leakage in a pH dependent manner at concentrations ranging from 3"5 to 52.0 pM. These effects were specific for negatively charged phospholipid vesicles. The peptide was also able to haemolyse erythrocytes. This study supports the notion that the sequence might be important in the initial infective steps of this virus, interacting with the target membranes and bringing about their subsequent destabilization.

Based on the predicted capacity to interact with membranes at the interface, we have found three regions in the ectodomain of the hepatitis C virus envelope glycoprotein E2 (430-449, 543-560 and 603-624) with the ability to destabilize membranes. Three peptides corresponding to the sequence of these regions have been synthesized and their interaction with liposomes have been characterized. The three peptides were able to insert deeply into the hydrophobic core of negatively charged phospholipids as stated by fluorescence depolarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Peptides E2(430-449) and E2(603-624) were able to induce aggregation of phosphatidylglycerol vesicles in a concentration-dependent manner both at neutral and acidic pH while peptide E2(543-560) did not induce any increase of optical density at 360 nm in the concentration range studied. The three peptides induced lipid mixing and the release of the internal contents in a dose-dependent manner when acidic phospholipids were used. Fourier transformed infrared spectroscopy indicated that the peptides adopted mainly a beta-sheet conformation which is not modified by the presence of acidic phospholipids. Taken together, our results point out to the involvement of these three regions in the fusion mechanism of HCV at the plasma membrane level.