Caveolae a new subcellular transport organelle

Recent advances have allowed the identification and characterization of well defined vesicular subcellular organelles involved in multiple basic cellular physiological processes, with demonstrated clinical relevance. Among these, three particular subcellular organelles have received special attention based on their proven and postulated participation in the sorting and targeting of small-and large-molecular weight molecules during exocytosis and endocytosis, and in cell signaling and transduction events. These have characteristic proteinaceous coat structures that allows their classification accordingly, into what has been described as clathrin coated vesicles and COP-coated vesicles and caveolae. In this review article a brief description of clathrin-coated vesicles and COP-coated vesicles is presented. Caveolae (CAV), in turn, constitute a novel subcellular organelle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling and transduction events. In this review of the literature a more extensive discussion is presented of CAV. In this context the article discusses the structural features of caveolae, its constituent protein caveolin(s), the functional aspects of this new organelle, and its postulated clinical relevance

Vesicular transport: implications for cell polarity

In polarized cells intracellular sorting of plasma membrane proteins occurs to a large extent at the trans-Golgi network, giving rise to vesicles destined for distinct plasma membrane domains. This review discusses the several pathways, both direct and indirect, which lead to protein incorporation into the correct cell surface, as well as the mechanisms involved. Proteins contain signals which direct their incorporation into the distinct vesicles destined for plasma membrane microdomains. Specific coat proteins are involved in vesicle assembly and are likely to play a role in the generation of discrete vesicle populations. Molecules involved in vesicle docking and fusion may also add specificity to the targeting process.

Calcium- and zinc-binding proteins in intracellular transport

The complex mechanism of intracellular transport is regulated by free calcium in different manners. Calcium binding proteins regulate several aspects of the vesicle fusion mechanism mediated by NSF (N-ethylmaleimide sensitive fusion factor). At least in some regulated exocytosis, calcium-binding proteins are the trigger for fusion downstream of NSF, Still, calcium-binding proteins, such as annexins, may be part of a different fusion mechanism mediating some specific transport steps or working in parallel to the NSF-dependent fusion process. Calcium is not the only ion necessary for the function of factors involved in vesicular transport. A zinc requirement has been also proposed. One of the zinc-dependent factors is probably a protein with a cysteine-rich region that coordinates zinc and binds phorbol esters. Although protein kinase C is the more prominent family of proteins carrying this domain, the factor necessary for transport does not appear to function as a kinase.

Mechanism of formation of post Golgi vesicles from TGN membranes: Arf-dependent coat assembly and PKC-regulated vesicle scission

We have developed an experimental system that utilizes purified Golgi fractions obtained from virus infected infected MDCK cells to reproduce in vitro the process of vesicle generation in the trans Golgi network, an important site for the sorting of proteins addressed to the plasma membrane, secretory vesicles, or lysosomes. Using an integrated biochemical and electron microscopic approach, we have shown that the formation of post Golgi vesicles carrying proteins destined to both plasma membrane domains of epithelial cells requires the activation of an ArF-like GTP-binding protein that serves to promote the assembly of the protein coat necessary to deform the donor membrane and generate a vesicle. The formation of the post Golgi vesicles also requires the participation of a Golgi membrane-associated Protein Kinase C, but not its phosphorylating activity. Other authors have shown that this is also the case for the PKC activation of the enzyme phospholipase D, which generates phosphatidic acid from phosphatidyl choline and may be involved in remodeling of membranes. We have been able to dissect the process of post Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and a subsequent one of vesicle scission. The first stage can occur at 20 degrees C and requires the activation of the Arf protein necessary for coat assembly. The second stage does not require nucleotides or an energy supply, but requires cytosolic proteins, and in particular, an NEM sensitive membrane scission promoting activity that operates only at a higher temperature of incubation. Because various PKC inhibitors blocked vesicle scission without preventing bud formation, we propose that the PKC is required for the activation of a PLD in the TGN, which leads to remodeling of the donor membrane and the severing of connections between the emerging vesicles and the membranes.