Cisternal Migration

Hank Hymanson

Supporting Evidence for the Theory of Cisternal Maturation-Progression
Abstract
Two competing theories have been postulated for explaining how proteins traverse the Golgi: cisternal maturation-progression and vesicular transport. Cisternal maturation-progression postulates that proteins move through the Golgi within cisternae which progress from cis to medial to trans. Large protein aggregates have been shown to move through the Golgi in a manner consistent with cisternal maturation-progression. COPI vesicles act as retrograde transport vehicles and have been shown to incorporate resident Golgi enzymes. A theory of competition among resident Golgi enzymes has been proposed to explain the polarity and specific distribution of enzymes throughout the Golgi, although further investigation is needed. Individual cisternae have been observed using time-lapse 3D microscopy to contain cis, medial, and trans resident Golgi enzymes progressively. Pulse-chase experiments have been coupled with the time-lapse 3D microscopy to show that cisternal maturation-progression can account for the rapid secretion of certain proteins. Further investigation is needed utilizing various cellular systems.
Background
How do proteins traverse the Golgi apparatus? This question is the basis of a fundamental argument regarding the origin, development and function of the Golgi. The Golgi is an essential organelle that is intricately involved in the transport and modification of proteins. Most proteins are synthesized within the endoplasmic reticulum, then transported to and modified sequentially within the cis, medial and trans Golgi. This organelle is highly involved in both the endocytic and exocytic pathways and as such is essential for cell survival. There are two distinct theories that prevail for explaining how proteins move through the Golgi: the theory of vesicular transport and the theory of cisternal maturation-progression. Vesicular transport postulates that the cis, medial and trans Golgi are static entities and that proteins are transported forward from one region to the next via transport vesicles that bud from the rims of each static cisternae. In contrast, cisternal maturation-progression states that rather than being static entities, Golgi cisternae are dynamic structures that originate from vesicles in the endoplasmic reticulum, accumulate to form cis cisternae, then progress sequentially from cis to medial to trans cisternae and eventually the trans cisternae bud off into various transport vesicles. Currently both of these theories are supported by distinct groups of researchers and as such no consensus exists regarding protein trafficking through the Golgi apparatus. The purpose of this paper is to present supporting evidence for the theory of cisternal maturation-progression.
Prior to delving into the specifics regarding vesicular transport and cisternal maturation-progression, it is important to establish the basic mechanisms associated with both of these theories. Coats are spherical protein shells with fixed compositions made up of multiple copies of cytosol derived subunits (coatomers) (Rothman & Wieland 1996). The two types of coated vesicles discussed in this review are COPI and COPII. The physical difference between COPI and COPII vesicles are the subunits that are included within each of these coats. COP coats are attached to budding membranes via the GTPase ADP-ribosylation factor 1 (ARF1). The COP coat is assembled when ARF1 is bound to GTP. When ARF1 hydrolyzes GTP to GDP the COP coat disassembles, thereby allowing the vesicle to dock with the corresponding acceptor membrane. Vesicle targeting and docking is mediated by a class of proteins known as Soluble NSF Attachment Protein Receptors (SNAREs). The vesicles contain v-SNAREs and the acceptor membranes or target membranes contain t-SNAREs. v-SNAREs are covered by the COP coat and therefore vesicles cannot attach to their target membranes until the vesicles have shed their coat. Another important component of this transport machinery are KDEL receptors. KDEL receptors are used to tag ER proteins for retrieval from the Golgi. As such, these receptors are essential for identifying retrograde vesicles.
The Golgi was first discovered in 1898 and first visualized using an electron microscope in 1954, however fundamental questions regarding the functioning of this essential organelle remain unsolved. (Martinez-Menarguez et al. 2001). From the 1960’s to the mid-1980’s the theory of cisternal maturation was embraced by most scientists within the field. This was mainly due to electron microscopy studies conducted on various species of plants and algae which found that large ER-derived products traversed the Golgi without being incorporated into any sort of transport vesicle. Some of these studies will be evaluated later in this paper. In the mid 1980’s numerous studies associated anterograde transport of cargo proteins with transport vesicles known as COPI vesicles. These discoveries allowed the theory of vesicular transport to gain considerable prominence over cisternal maturation-progression. However, in 1994 Cosson & Letourneur made the shocking discovery that COPI vesicles mediate retrograde Golgi to ER transport. This discovery was shocking because COPI vesicles were the only vesicles observed to bud within the Golgi stack (Pelham & Rothman 2000). If all COPI vesicles are assumed to be functionally the same, the fact that COPI vesicles have been observed moving in the retrograde direction calls into question the theory of vesicular transport. Thus, this discovery was the beginning of the current debate existent within the field regarding the true nature of intra-Golgi transport. It is well established that COPII coated vesicles form within the ER and transport proteins to the endoplasmic reticulum intermediate compartment (ERGIC). The key issue within the debate between vesicular transport and cisternal maturation-progression is whether COPI vesicles are involved in anterograde transport. Evaluation Some of the earliest evidence supporting the theory of cisternal maturation-progression comes from studies using algae. Algae are known to produce large cell-surface associated structures known as scales that are secreted via exocytosis (Melkonian et. al 1991). These scales are derived from the ER and are found in every layer of the Golgi. Numerous studies have shown that these scales are located exclusively within the Golgi cisternae and not within any vesicles outside of the Golgi (Brown 1974, McFadden & Melkonian 1986, Melkonian et. al 1991). This finding has been consistent within different types of algae, including flagellar and thecal. The fact that these large, ER-derived structures are able to reach the cell surface without being incorporated into any vesicles to traverse the Golgi provides strong evidence of cisternal maturation-progression in algae. Another example of a large macromolecular complex progressing through the Golgi without being incorporated into transport vesicles is procollagen I (PC). PC is a long, rod-like protein composed of three chains with over 1,000 amino acids. It is ideal for testing the maturation-progression model because it a) is decidedly larger than transport vesicles b) it has a molecular structure which precludes disassembly into smaller components that may be packaged into vesicles c) it is suitable for immunoelectron microscopy studies and biochemical quantition d) it its readily visualized using electron microscopy and e) its movement through the Golgi can be synchronized (Bonfanti et. al 1998). In order for PC to be transported from the ER to the Golgi it must be properly folded by the enzyme prolyl hydroxylase. Prolyl hydroxylase can be selectively and reversibly inhibited by an iron chelator referred to as DPD. Using DPD in ET-fibroblasts, Bonfanti and colleagues were able to synchronize the exit of PC from the ER and thus accurately track the movement of PC through the Golgi. Using various protocols Bonfanti and colleagues consistently observed that PC progressed from cis to medial to trans Golgi without leaving the lumen of each cisternae. This study (Bonfanti et. al 1998) provides further evidence for the cisternal maturation-progression model. As stated previously, the key issue in the debate between vesicular transport and cisternal maturation-progression is whether or not COPI vesicles are involved in anterograde transport. Although there is a consensus within the field that COPI vesicles are involved in retrograde transport those that support the theory of vesicular transport believe that two distinct populations of COPI vesicles exist. In an early study, COPI vesicles were found to contain the anterograde transport marker VSV-G (Orci et. al 1986). Following the discovery and subsequent acceptance that COPI vesicles are definitively retrograde vesicles another study was conducted which found two distinct populations of COPI vesicles, one retrograde and one anterograde (Orci et. al 1997). This study was conducted using immunoelectron microscopy upon both whole cells and a cell-free system. This study found both KDEL (retrograde marker) and VSV-G (anterograde marker) within every level of the Golgi and within COPI vesicles. Interestingly, no significant co-localization was observed between these two proteins. Based on this finding, it was postulated that two distinct populations of COPI vesicles exist; one that is responsible for retrograde traffic and one that is responsible for anterograde traffic. Another study was conducted which took into account resident Golgi enzymes, anterograde cargo and recycling proteins and their potential association with COPI vesicles. Resident Golgi enzymes (ManII, GalT, NAGT I, GalNAcT) were found to be incorporated within COP I vesicles, however at comparably lower concentrations than in the Golgi cisternae they were derived from (Sonnichsen et. al 1996). In addition, the anterograde proteins PIgR and RSA were observed to be incorporated into COPI vesicles, but again at comparably lower concentrations than in the Golgi cisternae (Sonnichsen et. al 1996). The ER-recycling proteins KDEL receptor and p58/ERGIC 53 were also observed within COPI vesicles at substantially higher rates than both the retrograde and anterograde cargo. This is consistent with the notion that KDEL receptors and p58/ERGIC 53 are part of the machinery for assembling COPI vesicles. In addition, an important detail that must be included in the description of this study is the fact that GTPγS was used prior to quantitating the incorporation of various proteins into COPI vesicles. Although Orci et. al’s 1997 paper seemed to provide a compelling argument for the theory of vesicular transport, and Sonnichsen et. al’s 1996 paper seems to somewhat support this theory, a closer look at the methodology used within each of these studies reveals a critical flaw. This flaw is described in detail by Nickel et. al 1998. Prior to Nickel’s 1998 paper, most studies attempting to investigate the nature of intra-Golgi transport utilized the mutated non-hydrolyzable form of GTP, GTPγS. GTPγS is generally used to accumulate abnormally large amounts of COPI vesicles. Interestingly, it was shown that the packaging of genetically engineered anterograde and retrograde cargo into COPI vesicles was significantly reduced by nearly a factor of 50 when GTPγS was used (Nickel et. al 1998). This finding indicates that uptake of cargo into COPI vesicles requires hydrolysis of GTP. In addition, it was found that KDEL receptor, p23 and p24 (transmembrane proteins associated with COPI formation) and ARF all increased in concentration when GTPγS was used. This identifies p23, p24 and KDEL receptor as part of the machinery needed for COPI vesicle formation (Nickel et. al 1998). One of the major conclusions from this paper was that COPI coated vesicles formed in vitro in the presence of GTPγS might not accurately represent physiologically loaded carriers. As such, the findings of Nickel et. al 1998 call into question the validity of the discovery of two separate populations of COPI vesicles by Orci et. al 1997 in addition to the low concentrations of cargo proteins observed within COPI vesicles in Sonnichsen et. al 1996. Thus far this review has provided background information for how intra-Golgi transport is postulated to operate, given early evidence for cisternal maturation-progression, provided counter-evidence in support of vesicular transport, and highlighted a critical flaw in the methodology of the studies that claim to support vesicular transport. For the remainder of this review various discoveries directly supporting cisternal maturation-progression will be discussed. Fundamentally there are two main questions which will be addressed: 1) Are anterograde cargo really incorporated within COPI vesicles? and 2) How does the Golgi maintain its highly polar nature and maintain localization of Golgi enzymes within specific regions (cis/medial/trans) of the Golgi? Although Orci et. al. 1997 claimed that two distinct populations of COPI vesicles exist, other studies conducted by other groups have only found one distinct population of COPI vesicles. For example, the previously discussed Procollagen I study (Bonfanti et. al 1998), failed to identify a significant quantity of anterograde cargo within any COPI vesicles. Another study, focused on COPI vesicle sorting under interphase and mitotic conditions (Sonnichsen et. al 1996), also failed to readily identify a significant population of anterograde cargo within COPI vesicles. Even more convincingly, in a study conducted within liver cells, known for their high level of secretory protein synthesis, anterograde proteins were not found to be significantly incorporated into COPI vesicles (Dahan et. al 1994). Collectively, these studies seem to indicate that COPI vesicles are involved solely in retrograde transport. The flawed methodology (utilization of GTPγS) employed within Orci et. al’s 1997 study could explain why their group is the only one to have identified two distinct population of COPI vesicles. If the theory of cisternal maturation-progression is truly the manner in which intra-Golgi transport is conducted, it must have a mechanism in place to account for the polarization observed across the Golgi and the localization/concentration of particular enzymes within different regions of the Golgi. If cisternae are constantly “maturing” outwards in the cis to trans direction there must be some sort of mechanism whereby resident Golgi enzymes are transported in the trans to cis direction. The best candidate for such retrograde transport of Golgi resident enzymes are COPI vesicles. Although Sonnichsen et. al 1996 did find certain resident Golgi enzymes to be located within COPI vesicles, the concentration of these enzymes was not sufficient to readily account for the type of retrograde transport that would be necessary to maintain an acceptable gradient to support the theory of cisternal maturation-progression. However, as demonstrated in Nickel et. al 1998, this observation could have been due to the fact that GTPγS was used in the process of obtaining this data. Another study, conducted without GTPγS found Golgi resident enzymes, but not the typical anterograde marker VSV-G to be incorporated within COPI vesicles (Love et. al 1998). This study was conducted by subjecting cellular homogenates, not treated with GTPγS, to velocity sedimentation on sucrose density gradients. In this study, the Golgi resident enzymes GalT and NAGT were found in two distinct populations of isolated membrane; one corresponding to COPI vesicles and one corresponding to Golgi cisternae whereas VSV-G was only localized in the fraction corresponding to Golgi cisternae. This study clearly indicates that COPI vesicles are involved in retrograde transport of Golgi resident enzymes. Another study, published only a year after the aforementioned sucrose density gradient paper, further indicates that COPI vesicles are involved in retrograde transport of resident Golgi enzymes (Lanoix et. al 1999). This study utilized a novel in vitro budding assay that allowed COPI vesicles to be formed via GTP hydrolysis. This new in vitro budding assay was adapted from the sucrose density gradient used in Sonnichsen et. al 1996 and allows for uncoated COPI vesicles to be detected along with coated COPI vesicles. Given what has already been reported about GTPγS (which locks vesicles into the coated conformation), this new budding assay appears to be a much more accurate representation of how COPI vesicles truly function. A major limitation associated with both the Orci et. al 1997 and Sonnichsen et. al 1996 papers was their focus only on coated COPI vesicles. As previously discussed, COPI vesicles need to shed their coat in order to allow binding to target membranes. As such, focusing only on coated COPI vesicles, as was done in both of the aforementioned studies, limited the scope of COPI vesicles that were observed. Using this new in vitro sucrose gradient technique, it was discovered that the Golgi resident enzyme α1,2-mannosidase II (manII) was concentrated nearly ten times greater within COPI vesicles then within the donor membrane (Golgi cisternae) (Lanoix et. al 1999). Interestingly, the concentration of manII incorporated within COPI vesicles declined significantly when a mutant, hydrolysis-deficient, GTPase arf-1 was introduced into the cells. This result is consistent with the finding in Sonnichsen et. al 1996, and indicates that the cell sorting process is mediated by the small GTPase arf-1. Furthermore, this same study found the level of the anterograde cargo protein polymeric IgA receptor found within COPI vesicles was negligible regardless of whether the cells were able to perform GTP hydrolysis (Lanoix et. al 1999). Collectively, the results from this study indicate that COPI vesicles do in fact transport resident Golgi enzymes in a GTP hydrolysis dependent manner. Additionally this study provides further support for the belief that anterograde proteins are not incorporated within COPI vesicles. Yet another study, conducted utilizing quantitative immune-EM on NRK cells, found resident Golgi enzymes but not anterograde cargo proteins to be incorporated within COPI vesicles (Martinez-Menarguez et. al 2001). This study found high levels of manII, the Golgi resident enzyme previously described in association with Lanoix et. al 1999, to be incorporated within these NRK cells. In addition, the COPI machinery proteins giantin, KDEL receptor and rBet1 were found to be significantly incorporated within COPI vesicles. In contrast, the previously discussed anterograde cargo protein VSV-G was not found to be significantly incorporated within COPI vesicles. This study utilized different methodology than either Love et. al 1998 or Love et. al 1999 and therefore provides further support to the finding that resident Golgi enzymes are incorporated into COPI vesicles and anterograde cargo proteins are not.
Although studies such as Love et. al 1998, Lanoix et. al 1999 and Martinez-Menarguez et. al 2001 indicate that resident Golgi enzymes can be incorporated into COPI vesicles, they do not fully explain the distinct localization of resident Golgi enzymes within various regions of the Golgi (cis/medial/trans). However, a mechanism has been proposed that seems to explain the asymmetric distribution of enzymes across the Golgi. This theory postulates that competition among enzymes for inclusion into COPI vesicles readily accounts for the differential distribution of enzymes throughout the Golgi (Glick et. al 1997). This theory states that different resident Golgi enzymes have different efficiencies for being incorporated within COPI vesicles, with cis-Golgi enzymes acting as the strongest competitors and trans-Golgi enzymes working as the weakest competitors. In various simulations, this theory was tested and it was established that the competition theory for asymmetric enzyme distribution holds true if most of the vesicles travel by one cisternae in the retrograde direction, as is postulated by the theory of cisternal maturation-progression (Glick et. al 1997). Although more comprehensive tests need to be conducted with known cis, medial and trans Golgi enzymes to verify this theory, it seems adequate to explain the relatively specific distribution of resident Golgi enzymes within different regions of the Golgi. Another consideration that must be taken into account if the theory of cisternal maturation-progression is going to be truly supported is the time it takes for a given ER-derived protein to pass through the various layers of the Golgi. In general protein secretion through the Golgi is considered to occur fairly rapidly (within only a few minutes). Conversely, it has been hypothesized that cisternal maturation-progression takes much longer to progress from cis to medial to trans. In 2001, an interesting study was published comparing the rate of processing of procollagen I and VSV-G (Mironov et. al 2001). In this study, human fibroblast cells were made to express both VSV-G and PC at high concentrations. These cells were then subjected to various synchronization protocols and the rate of processing through the Golgi was measured via electron microscopy and video. Interestingly, it was found that the PC traversed the Golgi at the same exact rate as the VSV-G. Furthermore, it was demonstrated that neither the VSV-G nor the PC left the lumen of the cisternae and, in fact, the two co-localized within the cisternae. Given the rest of the information already presented in this review, this paper provides very strong evidence for the accuracy of the theory of cisternal maturation-progression. Two more studies, published in the same volume of Nature in 2006 provide even further evidence for the theory of cisternal maturation-progression. One study used high speed three-dimensional confocal microscopy to observe the activity of fluorescently labeled cis (Sed5) medial (Gos1) and trans (Sec7) resident Golgi enzymes over time within the yeast Sacharomyces cerevisiae (Matsuura-Tokita et. al 2006). Interestingly, this study showed that, within any given cisternae, the label attached to the cis enzyme gradually faded and the label attached to the medial enzyme slowly became more prominent. Additionally, other experiments comparing the signal from tagged medial and trans resident enzymes showed that initially the medial enzyme (Gos1) had a strong signal that faded over time and the trans enzyme (Sec7) had a weak signal that built over time. This study (Matsuura-Tokita et. al 2006) provides compelling evidence that cisternae do, in fact, progress over time from cis to trans. In addition to the previously described study, another study was conducted using 3D time lapse fluorescent microscopy in combination with pulse chase experiments utilizing two distinct secretory proteins (Losev et. al 2006). Like the aforementioned Matsuura-Tokita et. al 2006 study, this study was conducted within the yeast Sacharomyces cerevisiae. For the 3D time lapse fluorescent microscopy Sec7 was used as a trans Golgi enzyme marker and Vrg4 was used as a cis Golgi enzyme marker. Each of these enzymes was fluorescently tagged and observed over time. Much like the Matsuura-Tokita et. al 2006 study, the cis marker started out with an intense signal that faded over time and the trans marker started with a faint signal that built over time. Overall, some fluorescence was observed within a given cisternae for four to six minutes (Losev et. al 2006). In order to better investigate whether the fluorescent transitions observed utilizing the 3D time lapse fluorescent microscopy actually represent the maturation kinetics of secretion traffic the timing of fluorescent observation of Vrg4 and Sec7 was compared to pulse chase data tracking the secreted proteins α-factor and carboxypeptidase Y. α-factor is one of the most rapidly secreted proteins in yeast and in this yeast it was observed to have half time of around seven minutes before it appeared in the extracellular medium. In addition, carboxypeptidase Y had a half time of roughly seventeen minutes before it appeared within vacuoles. The timing of the processing of these two established secretory proteins, when coupled with the observed 3D time lapse fluorescent microscopy indicates that the mechanism of cisternal maturation-progression is capable of processing secretory proteins in a manner consistent with their observed secretory timing. This paper provides the most compelling data for supporting the theory of cisternal maturation-progression. It not only physically shows individual cisternae maturing from cis to trans, it also takes into account the kinetics associated with secretory proteins and demonstrates that cisternal maturation-progression could truly be responsible for the movement of these proteins throughout the Golgi. Although not flawless and proven beyond a reasonable doubt, a considerable amount of evidence is available supporting the theory of cisternal maturation-progression over the theory of vesicular transport. As with any study, the studies reported within this review are subject to interpretation and each has their shortcomings. The theory of cisternal maturation-progression would be more definitively validated if more studies were done on various types of cells and if more studies were conducted testing the competition theory postulated by Glick et. al 1997 for explaining the differential distribution of resident Golgi enzymes throughout the Golgi. In addition, it is possible that different mechanisms exist within different systems for intra-Golgi transport. As such, it is conceivable that both the theories of cisternal maturation-progression and vesicular transport hold true within different systems. Although there is substantial room for further investigation and improvement into the theory of cisternal maturation-progression, this paper summarizes the abundance of research that is currently available which indicates that the theory of cisternal maturation-progression holds true within at least some systems.

Summary Within this paper, two of the prevailing theories for the nature of intra-Golgi transport have been discussed: cisternal maturation-progression and vesicular transport. The theory of cisternal maturation-progression first gained prominence in the 1960’s when it was found that in algae scales move through the Golgi without ever leaving the lumen of the cisternae. Adding credence to this theory, other large protein aggregates such as Procollagen I were also observed to traverse the Golgi without leaving the lumen of the cisternae. In the 1980’s the discovery of anterograde proteins within COPI vesicles allowed the theory of vesicular transport to gain significant prominence over cisternal maturation-progression. Then in a genetic study published in 1994 Cosson & Letourneur definitively associated COPI vesicles with retrograde transport. This discovery served as a contradiction to the vesicular transport theory, which had previously postulated that COPI vesicles were the main vesicles that anterograde proteins use to traverse the Golgi. As such, either the vesicular transport mechanism did not accurately describe how the Golgi functions or COPI vesicles are capable of both retrograde and anterograde transport. Although one particular group found two distinct populations of COPI vesicles (one anterograde and one retrograde), these findings were shown to be the result of flawed methodology (use of GTPγS) rather than accurate observation. If cisternal maturation-progression really is the mechanism whereby proteins produced in the ER are transported across the Golgi, an accurate mechanism must be in place to maintain the polarization and specificity of resident Golgi enzymes. This paper has shown that numerous papers have been published which show that resident Golgi enzymes are packaged into COPI vesicles for retrograde transport and that it is possible for the Golgi to maintain its polarization through the combination of anterograde moving cisternae (complete with cargo protein) with retrograde COPI vesicles (containing resident Golgi enzymes). The proposed competition theory for asymmetric distribution of resident Golgi enzymes throughout the cis, medial and trans Golgi was presented and hypothesized to accurately reflect how cisternal maturation-progression functions. Finally, 3D video microscopy was used to show that over time particular cisternae first contain a high level of cis resident Golgi enzymes that slowly fades and that same cisternae eventually comes to contain a high level of trans resident Golgi enzymes. Furthermore, the final paper discussed in this review correlates the timing observed using the 3D fluorescent microscopy with the results from pulse-chase experiments utilizing two distinct anterograde cargo proteins. This paper therefore provides evidence that cisternal maturation-progression is capable of moving anterograde cargo proteins through the Golgi at a rate consistent with the established short time frames associated with secretion of many secretory proteins. Collectively, the papers reviewed within this paper provide strong support for the applicability of the theory of cisternal maturation-progression in at least some cellular systems.

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