Reduction of the Golgi complex to it most basic components is a practical way to gain new insight into its functions. Herein we have established conditions for CHX treatment and demonstrate that these conditions effectively clear transmembrane and secretory proteins in transit from the Golgi complex. This reductionist approach has revealed basic properties inherent to the Golgi ribbon. First, the compact region of the ribbon is stable and lack of proteins in transit results in a reduction in diameter of all cisternae without changing their number. The noncompact regions appear less stable and seem to dissociate from the compact regions. Second, the compliment of cisternae remain the same and can be isolated with higher efficiency than the control. Third, proteins in transit account for approximately 50% of the protein in rat liver Golgi at steady state. Fourth, calcium transport into the lumen of the Golgi complex is mediated by two p-type calcium pumps. Finally, dissection of the proteins of the CHX-CHX SGF1 will lead to identification and understanding of the building blocks of this organelle.
The finding, that cis
-, medial-, trans
-, and TGN components of the Golgi complex remain in approximately the same proportions after CHX treatment has blocked entry of “new” proteins from the ER suggests that the Golgi is a very stable structure and that endogenous molecules are not degraded or mislocalized. Proteins in transit account for about 50% of the total protein of the Golgi fraction, based on the increase in enrichment of the CHX SGF1 over the CTL SGF1. This is in reasonable agreement with the earlier estimate of ~40% obtained by protein assay after high pH washing a Golgi fraction (Howell and Palade, 1982
). The difference in the two estimates can be partially accounted for by transmembrane proteins in transit, as high pH washing cannot remove this category of proteins from Golgi membranes. High pH washing has the following three disadvantages in preparing a Golgi fraction for identification of endogenous molecules: 1) transmembrane proteins in transit are not removed, 2) peripherally associated proteins (both luminal and cytoplasmic) that have unique Golgi functions are removed, and 3) enzymes may become inactivated. CHX treatment avoids all of these problems and is, therefore, an ideal method to use in the identification of the endogenous proteins of the Golgi complex. We have resolved the SGF1s by high resolution two-dimensional gel electrophoresis and the most abundant 173 Golgi specific proteins have been placed into three categories: cargo, cytosolic Golgi-associated, and resident Golgi proteins (Taylor et al., 1997
). We are working toward identifying already characterized proteins by using immunoblot analysis and identifying unknown species.
The fractionation scheme reported herein is a significant improvement over the 1970 protocol of Leelavathi et al. (1970)
; the stacked Golgi cisternae are separated from cytosol and other contaminating membranes increasing the enrichment 10-fold, resulting in a final enrichment of ~400-fold over PNS. In 1970 very few Golgi markers had been identified, so our evaluation of enrichment and yields of markers from the different subcompartments of the Golgi complex are clearly more complete. The enrichment of biochemical markers for cis
-, medial-, and trans
-Golgi in SGF1 isolated from CTL and CHX-treated animals is 300- to 700-fold. This is paralleled by an enrichment of morphologically defined Golgi stacks, cisternae, and vesicles of >90%. Strikingly, the morphometric analysis shows that the percent of vesicles observed immediately adjacent to Golgi stacks is reduced by half in the CHX fraction (7%) compared with CTL (13%). These data are consistent with the reduction in the enrichment of the coat proteins clathrin and β-COP from 23- and 35-fold in CTL SGF1 to 12- and 20-fold in CHX SGF1. Secretory proteins in transit, transferrin and apoE, are enriched 50-fold in the CTL SGF1 and could not be detected in CHX SGF1. These data demonstrate that secretory proteins have cleared the Golgi. This is dramatically corroborated by morphology. The cisternae of CTL SGF1 are wider and filled with lipoprotein particles. This is especially evident at the cisternal rims and what appear to be vesicles in the trans
region of the Golgi stack. After treatment with CHX, the cisternae are condensed and have reduced cisternal width, and lipoprotein particles are not evident within cisternae and vesicles surrounding the isolated stacks. Although there is no morphological correlate of transmembrane proteins in transit, the biochemical enrichment of HA4 and the pIgA-R shows these PM proteins enriched 16- and 110-fold, respectively, in the CTL SGF1, and both are effectively cleared from the CHX SGF1. A low level of contamination of the SGF1s determined by morphometric evaluation is borne out by the barely detectable levels of lysosomal and ER markers and their minimal enrichments in SGF1.
One noticeable difference in the Golgi marker enrichment data for both CTL and CHX fractions is that MG160, the medial marker, was found to enrich 150% more than p28 (cis) and TGN38 (trans). We attribute this difference to a greater stability and hence better recovery of the medial cisternae during the fractionation. The cis and trans cisternae are more susceptible to shear in the homogenization and fractionation procedures. However, both the cis and trans markers are recovered at similar levels. This level of enrichment and yields for p28 (204- and 400-fold enrichment and 51% and 46% yields for CTL and CHX SGF1s, respectively) and TGN38 (233- and 376-fold enrichment and 42% and 37% yields for CTL and CHX SGF1s, respectively) are certainly sufficient for studies of Golgi function.
One might wonder why we have spent our time and resources to develop a fractionation procedure using rat liver rather than using cultured cells. To address this concern, we have modified this fractionation protocol to obtain a stacked Golgi fraction from normal rat kidney (NRK) cells. The fractions obtained contained fewer stacked Golgi cisternae, greater contamination and the yield was extremely low, 50 μg of protein/3 × 107 cells (our unpublished results). Furthermore, studies of function would not be time and cost effective nor would it be practical to use such fractions to define endogenous proteins.
The clearing of proteins in transit by CHX treatment has revealed many properties of the organelle and allowed isolation of a stacked Golgi fraction enriched in endogenous proteins. Our characterization of calcium uptake indicates that these fractions are suitable for the study of endogenous Golgi proteins and functions. Two different calcium uptake activities are characterized in the CTL SGF1. The first activity, accounting for ~50% of the total, is consistent with a PMCA isoform(s). It is thapsigargin resistant, vanadate sensitive, oligomycin resistant and is cleared from the Golgi fraction after CHX treatment of the animals. A signal for PMCA was detected in CTL SGF1 but was not detected in the CHX SGF1 by immunoblot analysis. These data provide convincing evidence that the thapsigargin-resistant activity is not a resident Golgi activity and is most likely due to the PMCA isoform(s) in transit. Thus molecules in transit can function en route and contribute significantly to total calcium transport within the Golgi complex.
The other calcium uptake activity in the SGF1s corresponds to a SERCA class of intracellular calcium pumps. This activity is thapsigargin and vanadate sensitive, oligomycin resistant, and could not be distinguished from the ER calcium pump activity, in all experiments performed. The thapsigargin dose–response curve and IC50
values were the same for SGF1 and ER fractions in the presence of low and high free calcium levels. Immunoblots of the fractions, with an antibody that recognizes all isoforms of SERCA, revealed the same 110-kDa band in both the ER and SGF1s with enrichment levels of one- to fivefold. Upon further examination using autophosphorylation experiments, the only autophosphorylated band in the SGF1s that was EGTA sensitive and calcium stimulated was of the same molecular weight as the SERCA pump of the ER (110 kDa). The autophosphorylation was also thapsigargin sensitive. Wu et al. (1995)
suggest that SERCA2b is the only isoform expressed in liver and it is reasonable to expect this autophosphorylated band to correspond to a SERCA2b isoform.
These data argue that there is no biochemically unique resident calcium ATPase in the rat liver Golgi fractions. On the other hand, there is both SERCA protein and activity associated with the Golgi complex. Any Golgi-specific activity would be expected to enrich to the same level as the well characterized resident Golgi transmembrane proteins (200- to 400-fold). The much lower enrichment for SERCA results from it being distributed in two compartments, ER and Golgi. Because in rat liver hepatocytes, the ER is a large compartment containing approximately 50% of total cellular membranes, and the Golgi is a much smaller compartment, containing approximately 7% of total cellular membrane, a much lower level of enrichment for a protein equally distributed in both membranes is expected (Weibel et al., 1969
Data to suggest that the SERCA activity is actually in the Golgi and not a result of ER contamination come from two different experimental approaches. First is the morphometic and biochemical characterization of the fractions presented here. The ER contamination was at the level of 1%. Because both fractions had approximately the same uptake activity (pmoles of calcium per minute per microgram of protein), this would translate into the contaminating ER in the SGF1 transporting calcium at 100 times the rate it transports it in the ER fraction. Second is the data that in the presence of oxalate, calcium uptake was enhanced 40% in CTL and 60% in CHX ER. However, SGF1 calcium uptake showed no oxalate enhancement. This means that the membranes in the SGF1s containing the calcium uptake activity do not transport oxalate into the lumen of the Golgi fractions but the ER fractions have oxalate transporting activities.
What about the mammalian homologue of the yeast PMR1? Because PMR1 is localized to the Golgi, perhaps the mammalian homologue is as well. Too little is known at the present to draw reasonable conclusions and appropriate reagents are not avaliable to test its presence in rat liver fractions. If the PMR1 homologue is present in the Golgi fractions, it must be biochemically indistinguishable from the SERCAs.
We conclude that the Golgi complex does not contain a unique resident calcium transporting ATPase and that all calcium uptake into SGF1 can be attributed to two calcium uptake mechanisms: first, via a thapsigargin-resistant p-type pump that is not resident to the Golgi complex and corresponds to PMCA isoform(s) in transit to the PM, and second, SERCA pumps that are not restricted to the ER membrane. These two calcium pumps, along with any free calcium and/or calcium bound to soluble luminal proteins moving from the ER to and through the Golgi, supply the calcium required for the many functions of the Golgi complex.