THE definition of cellular organelles has evolved over the last hundred years largely driven by morphologic observations, but more recently has been supplemented and complemented by functional and biochemical studies (Palade, 1975) . Thus, organelles are now identified both by their morphology and by the set ofcomponents that comprise them . Determining how organelle identity is established and maintained and how newly synthesized protein and membrane are sorted to different organelles are the central issues of organellogenesis . Essential to the many cellular functions that take place within the central vacuolar system (which consists ofthe ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic which mediates the exchange of components between different organelles . There are two critical characteristics of membrane traffic . First, only certain sets oforganelles exchange membrane and the patterns of this exchange define what are called membrane pathways . Second, multiple pathways intersect at specific points within the central vacuolar system . For specific components to "choose" the correct pathway at such points of crossing, mechanisms exist to impose choices on specific molecules . This process is called sorting . The characteristicsofeachorganelle within the central vacuolar system are likely to be intimately tied to the properties ofmembrane traffic . An imbalance in the magnitude ofmembrane input into and egress from an organelle would have profound effects on the size ofthat compartment . In addition, failures in sorting or aberrations in targeting pathways would be expected to profoundly affect the identity of individual organelles . Recently, the relationship between the control of membrane traffic and the maintenance of organelle structure has been investigated with the use ofa remarkable drug, brefeldin A (BFA).' In this review we will summarize recent findings with BFA and propose some speculative models concerning the mechanism and regulation ofmembrane traffic within the central vacuolar system .

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Brefeldin A: insights into the control of membrane traffic and organelle structure.

Protein folding in the endoplasmic reticulum (ER) is monitored by ER quality control (ERQC) mechanisms. Proteins that pass ERQC criteria traffic to their final destinations through the secretory pathway, whereas non-native and unassembled subunits of multimeric proteins are degraded by the ER-associated degradation (ERAD) pathway. During ERAD, molecular chaperones and associated factors recognize and target substrates for retrotranslocation to the cytoplasm, where they are degraded by the ubiquitin-proteasome machinery. The discovery of diseases that are associated with ERAD substrates highlights the importance of this pathway. Here, we summarize our current understanding of each step during ERAD, with emphasis on the factors that catalyse distinct activities.

One step at a time: endoplasmic reticulum-associated degradation

Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. We have now visualized ER-to-Golgi transport using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP became concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocated inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurred during their translocation to the Golgi complex and they frequently stretched into tubular shapes. Together, our results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport.

ER-to-Golgi transport visualized in living cells

Cet article synthetise les connaissances sur le repliement et l'oligomerisation des proteines du reticulum endoplasmique: proteines membranaires, glycoproteines, proteines de liaison

Protein oligomerization in the endoplasmic reticulum

A combination of biochemistry in animal cell-free systems and genetics in yeast is revealing the molecular machinery of the secretory pathway of eukaryotes. Transporting vesicles have a simple coat structure and employ a general mechanism for fusion that is conserved in evolution.

Molecular dissection of the secretory pathway

Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex.

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.

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Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi.

ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments.

Publisher Summary This chapter discusses the procedures involved in the preparation of semiintact Chinese hamster ovary (CHO) cells, and the assay for measurement of transport of protein between the endoplasmic reticulum and the Golgi. The techniques are rapid and highly reproducible when applied to a healthy population of confluent CHO cells. For cells grown on tissue culture dishes, it is important that they are not overconfluent to obtain a high yield of semiintact cells using either technique. In all cases where the technology is extended to different cell lines, it will be important to optimize growth conditions to achieve appropriate levels of confluency, and to optimize conditions for swelling, or attachment of cells to the nitrocellulose filter. The ionic strength of the swelling buffer should be considered to be a critical determinant in the preparation of a population of semi-intact cells that can be perforated efficiently during scraping without release of intact cells or extensive cell disruption. Semi-intact cells can also be made from cells grown in suspension by swelling in a low osmotic strength buffer followed by a more gentle douncing protocol than that used to prepare cell homogenates.

D S Keller

Papers

Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex.

Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi.

ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments.

Preparation of semiintact Chinese hamster ovary cells for reconstitution of endoplasmic reticulum-to-Golgi transport in a cell-free system.