Registro de Inmobiliarias

The TGN is regarded as a specialized compartment involved in the sorting of proteins and lipids to their final destinations, and is the final exit point from the Golgi apparatus (Griffiths and Simons, 1986; Simons and van Meer, 1988). The TGN was originally defined as the compartment in which VSVG accumulated in a sialylated form on reducing the temperature to 20°C (Matlin and Simons, 1983; Saraste and Kuismanen, 1984; Griffiths et al., 1985). The TGN harbours the last two enzymes of the N-linked glycosylation pathway GalT and SialylT (Roth et al., 1982, 1985; Rabouille et al., 1995a), and the integral type I membrane protein TGN38 (Luzio et al., 1990), whose function remains obscure. However, in contrast to GalT and SialylT which are also present in the trans most cistema of the Golgi stack as well as the TGN, TGN38 is exclusive to the TGN. Tyrosine sulphation (Niehrs et al., 1994) and proteolytic cleavage o f viral glycoproteins and cellular protein precursors also occurs in the TGN (Sossin et al., 1990). The TGN may have a lower pH than preceding compartments in order to better facilitate these reactions (Anderson and Pathak, 1985; Seksek et al.,

1995; Kim et al., 1996; Llopis et al., 1998).

The TGN can be readily visualized by EM by labelling with thiamine pyrophosphatase (Novikoff, 1967), cytidine monophosphatase (Novikoff and Novikoff, 1977), and NBD-ceramide (Pagano et al., 1989, 1991) and exhibits a complex three dimensional tubulo-reticular morphology at the trans-fdiCQ o f the Golgi stack. The

Chapter 1________________________________________________________Introduction

TGN can be composed of as many as 1-6 superposed cisternal elements, which often do not remain strictly parallel to each other, but often ‘peel o ff from the Golgi ribbon and are continuous at their ends with tubules and condensing vacuoles (Ladinsky et al., 1994; Rambourg and Clermont, 1997). As with the CGN, a recent high voltage EM tomographic study on fast frozen, freeze-substituted NRK cells revealed the TGN not to be so reticular in nature, but more cisternal, suggesting that this morphology may be partly an artefact of fixation (Ladinsky et al., 1999). This study also revealed extremely close apposition of the TGN with the rER, which even wraps around trans-Go\gi

elements, with the ribosomes always on the side that is distal to the Golgi. This region of ER lacks vesicle buds suggesting there is no direct vesicular transfer between the compartments. The close apposition may facilitate the exchange of lipids between membranes by a nonvesicular mechanism (Ladinsky et al., 1999). Similar appositions between the ER and mitochondria are also seen and proposed to have this same function (Ardail et al., 1993).

The TGN displays a distinct response to BFA treatment relative to other Golgi compartments, in that it fails to redistribute to the ER and rather collapses onto the centrosome (Reaves and Banting, 1992) and even shows increased continuity with the plasma membrane (Lippincott-Schwartz et al., 1991). This emphasizes the discrete compartmental nature and membrane dynamics of the TGN relative to the rest of the Golgi apparatus.

The TGN serves as a sorting station interface of membrane flow between the Golgi apparatus, the plasma membrane and the endosome/lysosome system and can be seen as a directional valve between these systems (Mellman and Simons, 1992). Consistent with this is the fact that the TGN undergoes dramatic alterations in size and morphology contingent on the volume of membrane flow going through this compartment (Griffiths, 1989). The TGN is unique to the Golgi apparatus as it is the only site from which clathrin coated vesicles bud (Orel et al., 1984; Griffiths et al.,

1985), destined for either the plasma membrane or the endosomal system (Schmid, 1997). Mannose 6-phosphate receptors concentrate lysosomal destined hydrolases and

shuttle between the TGN and endosomes using clathrin coated vesicles in the TGN to endosome direction (Komfeld, 1992), and a novel class of coated vesicle containing TIP47 as a cargo selection device in the endosome to TGN direction (Diaz and Pfeffer, 1998). Secretory granules also form in neuroendocrine cells at the TGN (Tooze, 1998). A mysterious third population of vesicles with a Tace-like’ coat have also been observed budding from the TGN (Ladinsky et al., 1994). The sorting function of the TGN is perhaps best illustrated in polarized epithelial cells where the TGN must direct plasma membrane molecules to either the apical or basolateral domains of the plasma membrane. Basolateral targeting is often dependent on a signal in the cytoplasmic domain of the protein, and may be facilitated by a novel clathrin adaptor complex (Folsch et al., 1999). In contrast, apical targeting is thought to require segregation into glycolipid rafts which are selectively incorporated into apically destined transport vehicles (Simons and Ikonen, 1997).

Live cell studies that track the path of VSVG-GFP to the plasma membrane from the TGN have revealed that the primary vehicles responsible for this transfer were large (up to 1.5 pm in length) pleiomorphic tubulovesicular structures which bud as entire domains from the /r^zw-aspect of the Golgi apparatus (Hirschberg et al., 1998; Toomre et al., 1999; Polishchuk et al., 2000). These structures might be considered analogous to the VTCs that act as transport vehicles between the ER and CGN. They too undergo complex fusion and fission dynamics, and seem to move along microtubules (Hirschberg et al., 1998; Toomre et al., 1999; Polishchuk et al., 2000). Clathrin coated vesicles may even act to retrieve escaped TGN residents from these carriers, in a manner similar to the proposed retrieval of ftirin to the TGN from immature secretory granules (Dittié et al., 1997).

1.2 Principles of the secretory pathway.