PROTECCIÓN CATÓDICA

The Golgi apparatus is unique among the numerous intra-cellular organelles in that it consists of a stack of flattened cistemae which are held together as a cohesive unit. The rat liver Golgi preparation protocol (section 2.1.8) is sufficiently effective in preserving the stacked morphology of the Golgi, with 69% cistemae present in the stack (Fig. 3.3). In an investigation of stmctural components that could account for the proteinase sensitivity of Golgi stacks, uniform rectangular elements have been visualised between stacked Golgi cistemae (Amos and Grimstone, 1968; Franke et a l, 1972; Mollenhauer and Morre, 1975) and on dismpted, intact Golgi membranes using negative stain or tannic acid enhancement of positive stain (Cluett and Brown, 1992). These bridge-like stmctures were not artefacts because they were found both in vivo and in vitro, their dimensions between intact Golgi membranes were identical to those seen on single, unstacked Golgi membranes; they were removed following protease treatment under conditions that resulted in Golgi unstacking, and the heights of these elements were exactly the same as inter-cisternal space, i.e. ~11 nm. They could also be readily distinguished from clathrin coats and COP coats as they were much larger and wider than the bristles seen on clathrin-coated membranes (Pearse and Robinson, 1990) and were found on the planar faces of the cistemae, not on dilated rims or bud profiles where COP coats appear to form.

Biochemical investigations have led to the identification of a cytoplasmic matrix to which medial Golgi enzymes specifically bind (Slusarewicz et a l, 1994). This could represent the intercistemal material seen by EM (Cluett and Brown, 1992) and enhance the retention of Golgi enzymes and play a role in Golgi stacking since proteolysis of this matrix unstacks the cistemae. After sequential extraction of purified Golgi membranes with TX-lOO and salt, there are 10 major insoluble components in the matrix (Fig. 3.1). Using a combination of mass spectrometry and protein sequencing, some of these components were found to be cytoskeletal proteins (actin and cytokeratins) while others turned out to be contaminants present in the original Golgi preparation (rat uricase. Table 3.1). Work done in this laboratory has led to the identification of another component, GM130, a cw-Golgi matrix protein (Nakamura et a l, 1995). Other unidentified components might arise from matrices from different parts of the Golgi stack that are responsible for binding cis- and/or trans-Go\gi

Various aspects of the involvement of the cytoskeleton with the Golgi apparatus have been studied in different organisms. Microtubules are the major constituents of the cytoskeleton. They are involved both in these intracellular transport processes and in the spatial organisation of cytoplasmic organelles. The positioning of the Golgi apparatus has been shown to depend on an intact interphase microtubule network and perturbation of microtubules with exogenous agents such as nocodazole affected the integrity of the Golgi apparatus (Duden et a l, 1990; Kreis, 1990). The importance of m icrotubules in G olgi stacking has been studied in the fission yeast

Schizosaccharomyces pombe and it was shown that the disruption of the microtubule network can cause unstacking of Golgi cistemae(Ayscough et a l, 1993). Involvement of the actin filaments and intermediate filaments in Golgi structure and stacking is less well characterised.

One aspect of the involvement of actin and the Golgi was studied in the budding yeast

S. cerevisiae. Mutations in SA Cl, a gene identified by virtue of its allele-specific genetic interactions with yeast actin defects, were also capable of suppressing lethalities associated with yeast Golgi defects and sec9 (yeast SNAP-25 homologue, see section 1.3.5.1 and Table 1.2) secretory vesicle defects (Cleves et a l, 1989). These genetic data are consistent with the notion that the secretory pathway and actin cytoskeleton function was co-ordinated in the cell and both could be modulated by the same proteins. Secondly, Golgi-derived vesicles were shown to bundle actin filaments in an ATP-dependent manner via the mechanoenzyme myosin-I in the intestinal brush border (Path and Burgess, 1993). Lastly, in the slime mould Dictyostelium discoideum

alteration of the actin network led to the dispersal of the Golgi apparatus into vesicles distributed throughout the cell. A 24kD protein, comitin, that specifically binds F actin, was localised to the Golgi and proposed to link the Golgi apparatus to the actin network (Weiner et a l, 1993). However, actin's role in providing structural framework to the Golgi stack is less obvious. This is reflected in the finding that actin is not sensitive to chymotrypsin digestion at concentrations that unstacks the Golgi (Fig. 3.12). These evidence shows that actin is involved in vesicular transport and maintaining Golgi morphology, but does not necessarily show it is a component of the matrix.

The function of the cytokeratins in the Golgi matrix is currently uncharacterised. Their sensitivity to chymotrypsin at concentrations that precede Golgi unstacking indicates a possible role in providing a cytoplasmic link between Golgi proteins and the cisternal membrane, a function analogous to that of lamins.

Both actin and cytokeratins are involved in cytoskeleton anchoring to plasma membrane and cell adhesion. If they were involved in Golgi stacking, their importance will be difficult to assess beyond the current level using the protease digestion

approach. As cytoskeletal proteins are also involved in providing structural support to many other aspects of cellular function, genetic manipulations, e.g. mutation or knock­ out, will affect the cell as a whole and their specific contribution to Golgi stacking will thus be difficult to dissect from their general functions in other parts of the cell. An obvious way to test this is to do immuno-EM to see if they are present between stacked Golgi cistemae.

Following the proteolytic release from the stack, the individual cistemae maintained their flattened morphology and remained so for over an hour at 4°C. A 35kD protein was observed to be released into the supematant under conditions that precede Golgi unstacking. There are some indications that a protein of higher MW may be responsible for the release of p35 (Fig. 3.13), but due to technical difficulties in carrying out further identification using the eluted antibodies specific for p35, the identification of this higher MW protein was not completed. Immunofluorescence data suggest that p35 and/or the higher MW protein is in the Golgi apparatus (Fig. 3.14). Two possibilities exist: (i). p35 could be a degradation fragment derived from a higher MW component of the matrix that is sensitive to chymotrypsin digestion; or (ii). p35 could be an intact, chymotrypsin-insensitive protein itself but was released by the selective degradation of other anchor proteins. From the data available, these two possibilities could not be distinguished.