Function of stack.

Despite the evolutionary conservation of this elaborate stacked cistemal organization, its functional significance remains unclear and moot. However, a number of hypotheses have been proposed to explain the necessity of stacked, cistemal architecture.

The flattened disk shape of cisternal membranes has been suggested to contribute to the efficiency of glycosylation events that occur within the stack, by enhancing the surface area to lumenal volume ratio of the compartment. This would be advantageous if the glycosylation enzymes acted at a rate that was more rapid than the rate of sugar nucleotide import into cistemae. Increasing the surface area of the compartment relative to volume would allow these two processes to keep the same pace (Mellman and Simons, 1992). Alternatively, the high surface area to volume ratio o f cistemae may serve to keep secretory cargo concentrated to enable efficient glycosylation.

With regard to why these cistemae are stacked a number of hypotheses have been suggested. The first suggests the stack may be analogous to distillation tower, distilling secretory cargo away from escaped ER residents by an iterative process (Rothman,

Chapter 1_______________________________________________________ Introduction

1981; Rothman and Wieland, 1996). Since it is possible to retrieve ER residents even at the most /m w-pole of the Golgi stack the sorting of ER residents from secretory proteins may occur in a multistage process akin to fractional distillation (Rothman, 1981). Evidence for such a distillation process is provided by the fact that the cfr-Golgi cistemae have a higher freeze-fracture particle density than /rara-cistemae (Orci et al., 1998). However, since resident ER proteins rarely travel beyond the first two cistemae of the stack (Pelham, 1995), it would seem improbable that cells would require extended stacks just for the purpose of retrieving escaped ER residents. The extended stack may then be more relevant for the purposes of lipid sorting, which may be more difficult than protein sorting and so require multiple steps. Consistent with this is that cz5-Golgi (more ER like lipid composition) to trans-Qo\g\ (more plasma membrane like lipid composition) lipid gradients are observed (Orci et al., 1981; Van Meer, 1998). This would particularly apply to cholesterol, which must be rapidly pumped from its site of synthesis in the ER to the plasma membrane where it functions. Pumping the cholesterol out of the ER may be essential because high cholesterol levels may interfere with the translocation of nascent proteins across the ER membrane (Bretscher and Munro, 1993).

The stack may simply be an inescapable consequence of the mode o f transport through it and so represents a device that ensures rapid, efficient, processive vesiclular transfers between adjacent cistemae due to their close proximity (Palade, 1975; Orci et al., 1998; Sonnichsen et al., 1998; Shorter and Warren, 1999). However, this would not appear to be the case in yeasts. Firstly the majority of cistemae in S. cerevisiae are single (Preuss et al., 1992) suggesting that permanently stacked cistemae are not essential for efficient exocytic transport. However, stacks accumulate in certain mutant cells suggesting a stacking mechanism does exist (Novick et al., 1981; Benli et al., 1996). It may be that stacking is more transient in S. cerevisiae, enabling vesicle transfers only at specific times, or that vesicles are readily diffusible. Similarly, although S. pombe possesses a highly stacked Golgi apparatus, unstacking these cistemae using microtubule interfering agents, has no discernible effect on the rate of exocytic transport (Ayscough et al., 1993). However, the stacking mechanism in S. pombe may be distinct to that of

mammals in that microtubules are not required to maintain the mammalian Golgi stack, and unlike S. pombe the mammalian Golgi apparatus is not disrupted by inhibitors of protein synthesis (Ayscough and Warren, 1994). So far it has not been possible to unstack the mammalian Golgi apparatus, without inducing its vésiculation or tabulation, so similar manipulations of the Golgi have not been possible in mammalian cells. Until this is possible it is uncertain whether the Golgi stack contributes to exocytic efficiency per se.

Finally an ordered stack of cistemae may be necessary to perform efficient, sequential post-translational modifications of the secretory cargo as it moves en passant from cistema to cistema. This is best illustrated by the distribution o f N-glycosylation enzymes. These enzymes are distributed across the stack in the order in which they operate on the cargo passing through it, and can be considered analogous to an assembly line (Farquhar, 1985). Although this spatial organization of the glycosylation enzymes is not required for the correct execution of the sequential addition of sugar residues per se (Doms et al., 1989), it may well increase the efficiency of the process. Furthermore, if one adopts a cistemal maturation standpoint, one can then view the Golgi stack as a ‘delay timer’. By forcing a cistema to progress through the entire stack may ensure secretory cargo is exposed to the glycosylation machinery for sufficient time to allow correct modification with high fidelity (Glick and Malhotra, 1998). This may also explain the polymorphism of the number of cistemae per stack that exists between cell types in the same organism and between organisms. For it may be that the extent and complexity of the post-translational modification that must occur in the stack correlates positively with the number of cistemae per stack (Becker and Melkonian, 1996). For example, organisms such as ciliates which conduct little protein glycosylation and do not manufacture complex polysaccharides, tend to have few cistemae per stack. Whereas, the multifarious algal phyla synthesize massive quantities of highly complex carbohydrates and possess large numbers o f cistemae per stack (Becker and Melkonian, 1996).

Chapter 1_______________________________________________________ Introduction

Testing these hypotheses has proven extremely difficult since it is not yet possible to

precisely manipulate Golgi architecture, therefore evidence supporting such

hypotheses tends to be correlative rather than definitive. Manipulation of Golgi architecture requires a better understanding of the structural elements that underlie it.