Cell-free systems, vesicles studied

Vesicles lie at the heart of intracellular transport of many proteins. Recently, significant progress has been made in understanding the events involved in vesicle formation and transport. This has transpired because of the use of a number of approaches. These include establishment of cell-free systems with which to study vesicle formation. For instance, it is possible to observe, by electron microscopy, budding of vesicles from Golgi preparations incubated with cytosol and ATP. The development of genetic approaches for studying vesicles in yeast has also been crucial. The piemre is complex, with its own nomenclamre (Table 46-7), and involves a variety of cytosolic and membrane proteins, GTP, ATP, and accessory factors. 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

The complexity of the processes involved in plasma membrane biogenesis probably exceeds the possibilities of the mere in vivo analysis. In theory, cell-free systems should facilitate the study of the individual steps of the interaction between the various cell comparments. ROTHMAN et al. (11) provided the first evidence of the feasability of the approach and, recently, NOWACK et al. (12) described a cell-free system able to give informations on the budding from ER and the vesicle fusion with the GA. This system is suitable for the study of protein transfer, but could also be adapted to the case of the lipids. 

In our laboratory, studies of lipid transfer in leek seedlings in vivo, have already shown the existence of a vesicular process for the transfer of phospholipids and particularly of very long chain fatty acid-containing lipids [6]. This process follows the vesicular endoplasmic reticulum- Golgi apparatus- plasma membrane pathway. Using the cell-free system developed by Morre and coworkers, we have reconstituted in vitro the vesicular transfer of some phospholipids between the ER and the GA. This transfer is ATP and cytosol-dependent, is N Ethyl Maleimide and temperature sensitive and specific for the ER as donor and the GA as acceptor. The phospholipids transferred via an ATP-dependent manner in vitro between the ER and the GA were phosphatidylcholine (PC +79%), phosphatidylethanolamine (PE +67%) and phosphatidylserine (PS +123%) [7]. All those results are in favour of a vesicular transport of phospholipids between the ER and the GA of leek seedlings, and brought us to purify these transition vesicles issued from the ER. 

In addition to the vascular perfusion system studies, we have employed brush border membrane vesicles. Isolated from rat mucosa, to determine more closely the parameters of mucosal zinc transport (43). These vesicle preparations represent the best means currently available to delineate the characteristics of zinc transfer into mucosal cells. The technique permits isolation of the microvillous membrane free of other cellular contaminants, as determined by established procedures (44). 

Biotin uptake has been extensively studied in micro-organisms such as Lactobacillus plantarum, Saccharomyces cerevisiae, and Escherichia coli (30 and references cited). The micro-organisms are able to recover biotin from the medium and to concentrate it intracellularly by an active process, that is, against a concentration gradient, mediated by a specialized protein and dependent on an energy source. The biosynthesis of the transport system is regulated by the level of external biotin (30). Since biotin is not biosynthesized by mammalian cells, it must be obtained from exogenous sources by absorption. This uptake of free biotin has been studied, in relation to biotin deficiency, in the small intestine, liver, kidney, and placenta (reviewed in 31). Different models have been utilized (whole animal studies, everted sacs, brush border membrane vesicles. . . ), but the preferred model is cultured cell lines (31). It seems that biotin uptake in... 

---