Capítulo 5. Discusión de los Resultados
5.5 Relación entre las tareas que los profesores seleccionan y sus
In mammalian cells, p i 15 is a COPl vesicle tethering protein required for intra-Golgi transport in vitro and in vivo (Waters et ah, 1992b; Sonnichsen et ah, 1998; Seemann et ah, 2000). pi 15 is thought to contribute to the tethering process by cross-linking its two Golgi receptors: GM130 on the target membrane and giantin on the COPl vesicle. Given this apparent membrane cross-linking ability of p i 15, it has been tested whether p i 15 is capable of tethering cistemae to cistemae, thereby contributing to the stacking of Golgi cistemae, as well as playing a role in COPl vesicle tethering (Chapter 4; Shorter and Warren, 1999). This is conceivable given that giantin is present in cistemae and COPl vesicles at equal concentrations (Sonnichsen et ah, 1998). p i 15 is released from Golgi membranes during mitosis (Levine et ah, 1996), due to Cdc2 kinase mediated phosphorylation of GM130 (Nakamura et ah, 1997; Lowe et ah, 1998b), and this has been proposed to be a possible mechanism behind the accumulation of COPl vesicles at mitosis. However, if pi 15 also plays a role in the stacking of cistemae, does the release of pi 15 from the membrane also contribute to the mitotic unstacking process? During the disassembly reaction, it takes lOmin to deplete p i 15 from the Golgi membrane (Levine et ah, 1996). If this is the first stage of unstacking, then prior removal of p i 15 from the membrane, before entranee into the disassembly reaction, may accelerate the rate of unstacking.
85% of pi 15 can be removed from the Golgi membrane by IM KCl extraction (Waters et ah, 1992b; Levine et ah, 1996; see below). RLG were then either left untreated, extracted with 60mM KCl (which does not remove p i 15; Waters et ah, 1992b), or extracted with IM KCl and incubated at 37°C with mitotic cytosol for increasing time. At various times the reactions were fixed and processed for EM. Removal of p i 15 from the Golgi membrane by 1M KCl extraction does not change the percentage total membrane present as stacks (Figure 3.11 A, E and 1) which remains very close to 40% for all treatments, and is consistent with previous reports (Cluett and Brown, 1992; Hui, 1997). Thus, if p i 15 does contribute to cisternal stacking it is certainly not the sole determinant. On incubation with mitotic cytosol, IM KCl extracted RLG unstacked at an aecelerated rate compared to untreated or 60mM KCl extracted RLG
60mM KCl »
KCl ffo*
Figure 3.11 Effect of KCl extraction on the rate of mitotic unstacking of Golgi cistemae.
RLG were either left untreated, or extracted with 60mM KCl, or IM KCl and then incubated at 37°C with mitotic cytosol for increasing time, reactions were stopped at the indicated times, and processed for EM. (A-D) 0-20min time course for unextracted RLG. (E-H) 0-20min time course for 60mM KCl extracted RLG. (I-L) 0-20min time course for IM KCl extracted RLG. Large arrows denote stacks, small arrows denote
single cistemae. Note that stacks disappear and single cistemae appear more rapidly for IM KCl extracted RLG. Bar, 0.5pm.
Chapter 3__________________________________________ The Disassembly Reaction
(Figure 3.11, 3.12A). Unstacking was complete after just lOmin of the reaction for 1M KCl extracted RLG, whereas the same extent of unstacking took 20min in control reactions (Figure 3.12A). After just 5min of the reaction single cistemae were more apparent for IM KCl extracted RLG (Figure 3.1 IB, F, J; 3.12B), and by lOmin stacked stmctures were hard to find for IM KCl extracted RLG, yet made up c. 10% of the total membrane in control reactions (Figure 3.11C, G, K; Figure 3.12B). By 20min virtually no stacks remained in any of the incubations (Figure 3.1 ID, H, L; Figure 3.12A). The initial rates of loss of stacks (i.e. the rate of loss of stacks for the first 5min of the reaction) were 4.9%min’^ for untreated RLG, 4.8%min'^ for 60mM KCl extracted RLG and 6.2%min'^ for IM KCl extracted RLG. Suggesting that the initial rate of unstacking was 25% quicker for IM KCl extracted RLG. It was not determined whether the time point of 5min was still in the linear range, that is no earlier time points were looked at, therefore these differences in initial rate may actually be an underestimate. These differences did not appear to be due to any increase in the rate of consumption of cistemae which looked similar for all treatments (Figure 3.12C). In fact the initial rates of loss of cistemae (i.e. the rate of loss of cistemae in the first 5min of the reaction) were 3.7%min'* for untreated RLG, 2.9%min‘^ for 60mM KCl extracted RLG, and 2.3%min'^ for IM KCl extracted RLG. So even though the initial rate of unstacking was accelerated upon IM KCl extraction, the initial rate of loss of cistemae was actually reduced.
Obviously, IM KCl extraction of RLG will release numerous proteins other than p i 15 from the Golgi membrane (Figure 3.13A). In fact, 60mM KCl extraction removed numerous proteins from RLG as well, suggesting a number of loosely attached, possibly cytosolic proteins are present in the RLG preparation. However, a number of proteins Coomassie stained much stronger in the IM KCl supernatant as compared to the 60mM KCl supematant (arrows in Figure 3.13A), and these may be factors whose removal facilitates the more rapid unstacking. Given that p i 15 is very clearly removed from RLG by IM KCl and not 60mM KCl (Figure 3.13B) it was decided to supplement the IM KCl extracted RLG with purified rat liver p i 15 and determine whether this had any effect on the rate of unstacking.
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Figure 3.12 Quantitation of effect of KCl extraction on the rate of mitotic unstacking of Golgi cistemae. RLG were either left untreated, or extracted with 60mM KCl, or IM KCl and then incubated at 37°C with mitotic cytosol for increasing time, samples were processed for EM at various times and quantitated for the percentage total membrane present as stacks (A), single cistemae (B) and all cistemae (C). Values represent means±SEM (n=3).
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Figure 3.13 Effect of KCl extraction on Golgi membrane polypeptide composition. I0)ig RLG was either left untreated or extracted with 60mM KCl or IM KCl. After the extraction membranes were pelleted and the supematant (SN) removed. The membrane pellets were dissolved in SDS-PAGE sample buffer, and the polypeptides in the SN TCA precipitated, and then dissolved in SDS-PAGE sample buffer. Samples were then fractionated by SDS-PAGE using a 4-20% gradient gel, and either stained with
Coomassie (A) or transferred to nitrocellulose (B) and probed for pi 15 using the specific mAb 8A6. Molecular weights in kD are shown on the left. Arrows in A denote bands which appear stronger in the IM KCl SN as compared to the 60mM KCl SN. Asterisks in A denote soybean trypsin inhibitor which was used as a carrier protein for the TCA precipitation.
Excess p i 15 inhibits the consumption of cistemae during the disassembly reaction (Levine et al., 1996), so it was important to add back p i 15 to the level which it was present on RLG prior to IM KCl extraction. The levels of p i 15 on RLG, and the binding characteristics of p i 15 to IM KCl extracted RLG had been very carefully determined previously (Levine et al., 1996) and 0.1-0.15pg p i 15 was the maximum that could bind to lOpg IM KCl extracted RLG. Therefore, after IM KCl extraction, 0.075pg p i 15 was supplemented to 5pg IM KCl extracted RLG and allowed to bind for lOmin on ice. Membranes were then incubated at 37°C with mitotic cytosol for increasing time, and at various times fixed and processed for EM. Supplementing 1M KCl extracted RLG with p i 15 reduced the rate of cisternal unstacking (Figure 3.14, 3.15A), and returned it to levels similar to untreated RLG. The initial rate o f loss of stacks was 5.7%min‘^ without p i 15 supplementation and 4.5%min'^ with p i 15 supplementation. Stacks still represented c. 12% of the total membrane after lOmin of the reaction after pi 15 supplementation, but were virtually absent from unsupplemented IM KCl extracted RLG (Figure 3.14B, E). This was reflected in the changes in the percentage of total membrane present as single cistemae, which rose very sharply for 1M KCl extracted RLG in the first 5min of the reaction, but much less so when p i 15 was supplemented (Figure 3.14A, D; 3.15B). Again the increase in the rate of unstacking was not due to an increase in the rate of consumption of cistemae (Figure 3.15C). In fact the initial rate of loss of cistemae was slower for 1M KCl extracted RLG being 1.8%min'^ compared to 2.8%min'^ for IM KCl extracted RLG supplemented with pi 15. So again, even though the initial rate of unstacking was increased the initial rate of consumption of cistemae was decreased in IM KCl extracted RLG.
p i 15 can also be removed from Golgi membranes by incubating in the presence of N73pep, a peptide corresponding to the N-terminal 73 amino acids of GM130, which comprises the p i 15 binding site on GM130 (Nakamura et al., 1997; Shorter and Warren, 1999). Incubation of RLG for 15min on ice with 80pM N73pep is sufficient to remove 85-90% of the Golgi bound p i 15 (Figure 3.16A), and this should represent a more specific procedure to extract p i 15 from the Golgi membrane as compared to