- Email: [email protected]
The Golgi apparatus: going round in circles?
Research Update
TRENDS in Cell Biology Vol.12 No.3 March 2002
101
Research News
The Golgi apparatus: going round in circles? Francis A. Barr
Since the work of Palade and colleagues, protein transport from the endoplasmic reticulum (ER) and through the Golgi apparatus has been envisioned as a process primarily mediated by small vesicular carriers budding from one compartment and fusing with the next [1,2]. This view has been challenged in recent years by studies of the secretion of procollagen, a molecule simply too large to fit inside classical vesicular carriers of 60–70 nm diameter, which passes in an anterograde fashion across the Golgi stack without ever leaving the cisternae [3]. A cisternal maturation model was used to explain these data (Fig. 1). In such a model, Golgi cisternae arise by the fusion of vesicular or tubular intermediates from the ER and then mature from a cis- to a trans-form by the exchange of enzymes in small vesicular carriers moving in a retrograde fashion. Mironov and coworkers now extend their previous observations on procollagen to include the much smaller transmembrane glycoprotein of vesicular stomatitis virus (VSV-G) [4]. They find that neither cargo molecule enters transport vesicles while progressing through the Golgi stack, and these cargos are transported at the same rate, consistent with both being in the same maturing cisternal structure. Biochemical and morphological evidence supporting other aspects of the cisternal maturation model also exists, most importantly indicating that Golgi resident http://tcb.trends.com
PM
(a)
Golgi
TransMedial GTC
COP I
Cis-
COP I
rab1+p115 –
COP II
Sar1DN
ER
Recent studies have questioned the idea that the Golgi complex is a stable organelle with a unique identity through which secretory cargo is transported by vesicles. Instead, it is proposed that Golgi apparatus proteins continuously recycle via the endoplasmic reticulum by vesicle transport, whereas cargo molecules remain in maturing cisternal structures. Rather than forming a rigid matrix, structural Golgi proteins might be highly dynamic and recycle via the cytoplasm. I will discuss the evidence for these claims and consider whether or not they really disprove older ideas on how the Golgi apparatus is structured and performs its function.
(b)
Tethering complexes Enzymes Cargo TRENDS in Cell Biology
Fig. 1. A recycling model for Golgi traffic. (a) Golgi cisternae form by the fusion of transport intermediates arising from the COP II pathway. This can be blocked by dominant-negative forms of the Sar1 GTPase, which is required for the recruitment of COP II to endoplasmic reticulum (ER) membranes. The recognition and fusion of COP II vesicles involves the small GTPase rab1 and the tethering factor p115, plus a variety of other molecules needed to regulate rab1 activation [22]. Retrograde traffic between Golgi cisternae and back to the ER is mediated by COP I vesicles, and, within the Golgi, their tethering is likely to require the Golgi transport complex (GTC) [23,24] (b) Golgi enzymes recycle in COP I vesicles between adjacent cisternae and the ER. In this model, an important determinant of localization within the stack for resident proteins would be their rate of recycling in COP I vesicles – the lower the rate of recycling, the more trans the localization of the protein. These vesicles might also not be in free diffusion, being tethered by complexes such as the GTC or p115–GM130 from the moment of formation until reaching their destination.
proteins are sorted into recycling vesicles at the different levels of the Golgi stack [5–7]. Studies from the Nilsson lab describe the purification of different classes of COP I vesicles containing either cis- or medial-Golgi markers, but little anterograde cargo [6,8]. These vesicles select cargo by coupling the recruitment of
COP I complexes to GTP hydrolysis by the small GTPase ARF1. The mechanism for this appears to involve direct modulation of ARF1 GTPase-activating protein (GAP) activity by at least some transmembrane cargo molecules. The cytoplasmic domains of these proteins might inhibit the ARF-GAP activity,
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Research Update
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TRENDS in Cell Biology Vol.12 No.3 March 2002
(a)
Resident proteins Matrix proteins
(b)
Tethering/matrix complexes TRENDS in Cell Biology
Fig. 2. The Golgi matrix. (a) Matrix proteins such as GM130 might form an ordered structure, giving long-range organization to Golgi cisternae. Interactions of this structure with resident proteins such as enzymes would be important for their localization within the stack. (b) Alternatively, matrix proteins might function in much smaller units linking adjacent cisternae and serving to give compartmental identity during membrane traffic as part of tethering complexes. It is quite possible that both these proposals are correct, and different Golgi matrix proteins function in one or both of these ways.
allowing accumulation of active ARF1 on the membrane, thus promoting COP I recruitment and vesicle formation [8,9]. These findings that some, if not all, anterograde cargo is transported in maturing cisternae, while enzymes are present in COP I vesicles, support the maturation model for Golgi transport and therefore also raise issues concerning the maintenance of the stacked cisternal structure of the Golgi apparatus. A maturation model suggests that structural protein complexes, often referred to as the ‘Golgi matrix’, would also need to be highly dynamic, recycling as the cisternae mature, whereas previously they were considered to be relatively static components of long-lived cisternal structures. The Golgi matrix: is it dynamic or static?
The putative Golgi matrix was originally described biochemically and morphologically as a proteinaceous structure isolated from Golgi membranes that retained the form of Golgi cisternae http://tcb.trends.com
and was capable of specifically binding to Golgi enzymes (Fig. 2) [10]. Purified Golgi membranes retain their characteristic stacked cisternal structure, and it was proposed that the Golgi matrix proteins formed an exoskeleton required to maintain this unique organization. Many components of this Golgi matrix are coiled-coil proteins, the best characterized of which are the cis-Golgi proteins GM130 and its interacting partner p115 [11,12]. GM130 is targeted to Golgi membranes through an interaction with GRASP65, a protein identified in an in vitro functional assay for factors required for the stacking of Golgi cisternae [13]. It is interesting to note that GRASP65 is a myristoylated protein and that many other proteins with this modification can switch between a membrane-bound and cytosolic form in response to a trigger such as GTP binding [13,14]. A recent study on the dynamics of a GFP-tagged form of GRASP65 in vivo found that it appears to rapidly recycle on and off Golgi membranes [15]. Whether or not it remains bound to GM130 in these
experiments is unclear, but previous biochemical studies have found that GM130 and GRASP65 interact with high affinity, making it likely that the complex as a whole is recycling [13]. This study also found that there was a cytosolic pool of GRASP65 – despite the fact that this was not observed in previous work on the GRASP proteins. Another recent paper investigating how cis-Golgi matrix proteins target to the Golgi showed that newly synthesized endogenous GRASP65 and GM130 bind rapidly and directly to the Golgi, and in these experiments there was no evidence for a cytosolic pool of the two proteins [16]. This obviously presents a disparity that is impossible to resolve at the present time and will require further investigation. How can these findings be explained? One possibility is that the GRASP65–GM130 complex actively recycles from Golgi membranes to the cytosol, there being only a very small pool in the cytosol at any one time. Understanding the mechanism of matrix protein targeting to the highly dynamic Golgi cisternae and how this is regulated will be important for resolving this issue. Finally, it should also be remembered that exchange of membrane-associated Golgi matrix subunits with a soluble pool does not in itself prove that the cisterna is changing its identity. Does the Golgi matrix exist?
It is often stated that ‘seeing is believing’, and, because a Golgi matrix has been visualized by microscopy, it might seem odd to question the existence of such a structure. As originally envisioned, this structure should comprise a stable assembly of structural proteins imparting organization and identity to Golgi membranes. Warren and coworkers, who found that a Golgi-like structure exists independently of protein transport from the endoplasmic reticulum, provided evidence for this rigid view of the Golgi matrix [10,17]. In their experiments, these matrix structures resembled a Golgi apparatus under the light microscope in vivo, but unfortunately, under the electron microscope, they lacked a clear cisternal organization [17]. Obviously, more than just a Golgi matrix is required to build a Golgi apparatus, although these data do support the idea of a Golgi matrix structure independent of the ER. Two other studies have re-examined this evidence using microinjection or
Research Update
overexpression of dominant-negative Sar1 mutants to block ER exit by the COP-II vesicle transport pathway [15,18]. In both cases, Golgi proteins redistributed to the ER, and the Golgi apparatus disappeared. The kinetics of relocalization were different for Golgi enzymes compared with structural Golgi matrix proteins; the latter redistribute more slowly, and this effect requires higher concentrations of dominant-negative Sar1 [18]. While these observations do not really argue against a Golgi matrix imparting structure to the Golgi apparatus, they do question the independence of such a structure from the ER. However, a caveat to this is that it does not prove that Golgi matrix proteins ever recycle back to the ER under normal conditions. A second issue is that it takes many hours for Golgi matrix proteins to redistribute to the ER in these experiments, and, at shorter times, no effect on Golgi matrix proteins such as GM130 and GRASP65 is observed under comparable conditions [16]. However, even this point is disputed as other studies find a rapid redistribution of Golgi matrix proteins in minutes under apparently similar conditions [15]. Rather than debate the existence of such a matrix, it is probably more useful to ask whether its potential components are important for maintaining the Golgi and what their functions might be. GM130 is known to interact both with Golgi Rab proteins and with the vesicle tethering factor p115 and to play a role in the formation of Golgi stacks in a cell-free assay [19,20]. A novel Golgi matrix protein, golgin-45 was recently described as the binding partner for GRASP55 on the medial-Golgi [21]. Although the function of golgin-45 is not known, it binds to the small GTPase rab2, implicating it in some aspect of vesicle tethering to the medial-Golgi. Depletion of golgin-45 by RNA interference causes Golgi enzymes to collapse back into the ER, and other Golgi matrix proteins to redistribute to small punctate structures and the cytoplasm [21]. Not surprisingly this also resulted in a block to protein transport as there was no longer a functional Golgi apparatus. In summary, although the Golgi matrix proteins do seem to be important for maintenance of Golgi structure and to impart Golgi identity, the existence of an ordered structure justifying the term matrix is less clear. Rather than a static matrix, an alternative model should be http://tcb.trends.com
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considered in which these proteins exist in smaller landmark complexes specific for the different Golgi cisternae such as GRASP65–GM130–p115 on the cis-Golgi, and GRASP55–golgin-45 on the medial-Golgi to give compartmental identity (Fig. 2).
material from the ER to the plasma membrane? Currently, that there is a large recycling flux of resident proteins is the most popular explanation, but, as with many aspects of the Golgi, this is a circular argument subject to change. Acknowledgements
Concluding remarks
Because the secretory pathway is primarily a cargo transport system, it makes sense that small quantities of enzymes are taken to meet the large amount of substrates they need to modify. This would seem to be more effective than the converse situation, transporting large amounts of cargo in small vesicular structures. However, there are still some unresolved issues with this model. It has been reported that Golgi enzymes recycle via the ER and that all forward-moving cargo, presumably including enzymes, moves at the same rate through the Golgi apparatus. If this were the case, then the cis-Golgi would contain not only cis-enzymes but also significant amounts of recycled medial- and trans-enzymes on their way back to their correct locations. How then can their discrete distributions to the different layers of the Golgi be explained? The most parsimonious explanation is that a sequential enzyme recycling mechanism exists that transports enzymes to the preceding cisterna in vesicles, rather than allowing them to go directly back to the ER. If these vesicles were not in free diffusion but tethered to the adjacent destination compartment from the moment of formation, this could explain such a mechanism. To some extent, this is merely postponing the problem for a later date as the molecular details of cis-Golgi versus medial- or trans-Golgi localization for particular proteins still have to be explained. A second issue relates to the putative Golgi matrix proteins and how they are specifically targeted and then retain their localization to different cisternae within the stack. As discussed above, it is possible that some matrix proteins recycle via the cytoplasm, but others are integral membrane proteins and must therefore recycle in vesicles. Despite the shift in perspective from considering the Golgi apparatus as a static organelle to a more dynamic structure, the essential cell-biological question remains the same: how is this structure maintained in the face of a large flow of secretory
I thank Ben Short and Sean Munro for discussions during the course of writing this article and acknowledge the comments of the reviewers. References 1 Farquhar, M.G. and Palade, G.E. (1998) The Golgi apparatus – 100 years of progress and controversy. Trends Cell Biol. 8, 2–10 2 Rothman, J.E. and Orci, L. (1992) Molecular dissection of the secretory pathway. Nature 355, 409–415 3 Bonfanti, L. et al. (1998) Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell 95, 993–1003 4 Mironov, A.A. et al. (2001) Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae. J. Cell Biol. 155, 1225–1238 5 Love, H.D. et al. (1998) Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport. J. Cell Biol. 140, 541–551 6 Lanoix, J. et al. (1999) GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles. EMBO J. 18, 4935–4948 7 Martínez-Menárguez, J.A. et al. (2001) Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisterna-progression model of intra-Golgi transport. J. Cell Biol. 155, 1213–1224 8 Lanoix, J. et al. (2001) Sorting of Golgi resident proteins into different sub populations of COPI vesicles: A role for ArfGAP1. J. Cell Biol. 155, 1199–1212 9 Goldberg, J. (2000) Decoding of sorting signals by coatomer through a GTPase switch in the COPI coat complex. Cell 100, 671–679 10 Slusarewicz, P. et al. (1994) Isolation of a matrix that binds medial Golgi enzymes. J. Cell Biol. 124, 405–413 11 Fritzler, M.J. et al. (1993) Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex. J. Exp. Med. 178, 49–62 12 Nakamura, N. et al. (1997) The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Cell 89, 445–455 13 Barr, F.A. et al. (1997) GRASP65, a protein involved in the stacking of Golgi cisternae. Cell 91, 253–262 14 Goldberg, J. (1998) Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95, 237–248 15 Ward, T.H. et al. (2001) Maintenance of Golgi structure and function depends on the integrity of ER export. J. Cell Biol. 155, 557–570
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16 Yoshimura, S-I. et al. (2001) Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus. J. Cell Sci. 114, 4105–4115 17 Seeman, J. et al. (2000). Matrix proteins can generate the higher order architecture of the Golgi apparatus. Nature 407, 1022–1026 18 Miles, S. et al. (2001) Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block. J. Cell Biol. 155, 543–556 19 Shorter, J. and Warren, G. (1999) A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi
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cisternae in a cell-free system. J. Cell Biol. 146, 57–70 Weide, T. et al. (2001) The Golgi matrix protein GM130: a specific interacting partner of the small GTPase rab1b. EMBO Rep. 2, 336–341 Short, B. et al. (2001) A GRASP55–rab2 effector complex linking Golgi structure to membrane traffic. J. Cell Biol. 155, 877–884 Allan, B.B. et al. (2000) Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289, 444–448 Whyte, J.R.C. and Munro, S. (2001) The Sec34/35 Golgi transport complex is related to the exocyst,
defining a family of complexes involved in multiple steps of membrane traffic. Dev. Cell 1, 527–537 24 Walter, D.M. et al. (1998) Purification and characterization of a novel 13 S hetero-oligomeric protein complex that stimulates in vitro Golgi transport. J. Biol. Chem. 273, 29565–29576
Francis A. Barr Dept of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, Martinsried, 82152 Germany. e-mail: [email protected]
A phosphorylation-driven ubiquitination switch for cell-cycle control J. Wade Harper Cellular changes in state can be dictated by complex all-or-nothing switches built from ultrasensitive protein kinase cascades, positive-feedback loops and other mechanisms. Recent work has established that phosphorylation-driven protein destruction through the SCF ubiquitinligase pathway can also occur in a switchlike manner. In this context, multiple phosphorylation events are used to set a threshold for substrate targeting, thereby providing a framework for understanding the inter-relationship between protein phosphorylation and ubiquitin-mediated proteolysis.
The ability of complex cellular systems to undergo coordinate and irreversible changes in state is fundamental to the proper development of an organism. Such transitions frequently involve one or more molecular switches, capable of coordinately converting an entire pool of a crucial regulatory factor from one activity state to another over an extremely small time interval. Molecular switches often involve protein kinase cascades in which the response of the terminal kinase is ultrasensitive to the level of a continuously variable stimulus and is re-enforced by positive-feedback mechanisms [1]. In essence, the molecular circuitry inherent in an ultrasensitive pathway determines the threshold stimulus required for turning on the pathway in an all-or-nothing manner. Now new work [2] has established that phosphorylation-driven ubiquitin-mediated destruction of the cyclin-dependent kinase (CDK) inhibitor Sic1 may also occur in a http://tcb.trends.com
switch-like fashion during cell-cycle progression and, in so doing, sets the threshold for the level of upstream kinase activity required to turn on the destruction switch and activate DNA synthesis. In order to duplicate itself, a cell must make a copy of its genome through the process of DNA replication (S phase) and then segregate two equal copies of this genetic material into mother and daughter cells through the process of mitosis. The decision to enter into a new round of cell division is not taken lightly, and multiple control mechanisms are used to control the decision to move from the G1 phase of the cell cycle into the replicative stage. Entry into S phase in the budding yeast Saccharomyces cerevisiae requires Clb5–Cdc28 kinase, which is kept inactive during the G1 phase by the CDK inhibitor Sic1 [3]. During this period, cells sense environmental nutrients and convert these signals into G1 cyclin (Cln)–Cdc28 kinase activity (Fig. 1a). Once threshold levels of Cln–Cdc28 activity are achieved, Sic1 is rapidly phosphorylated, ubiquitinated and destroyed, thereby releasing Clb5–Cdc28 to activate S phase [4,5]. The necessity of properly controlling Sic1 destruction is revealed by the fact that cells lacking SIC1 begin DNA synthesis prematurely and display genomic instability. Sic1 ubiquitination occurs through the concerted action of E1 ubiquitinactivating enzyme, the E2 conjugating enzyme Cdc34 and an E3 ubiquitin ligase referred to as SCFCdc4 [6,7]. This ubiquitin ligase comprises Skp1, Cdc53/Cul1, Rbx1
(also called Hrt1 or Roc1) and Cdc4. Temperature-sensitive mutations in each subunit of SCFCdc4 cause G1 arrest and Sic1 stabilization in budding yeast. Skp1–Cdc53–Rbx1 forms a core ubiquitin ligase that interacts with E2s [8,9], while Cdc4, a member of the F-box family of proteins10, interacts with Skp1 through the F-box motif and with substrates through C-terminal WD40 repeats [7,10]. F-box proteins are a large family of proteins that generally serve as receptors for ubiquitination substrates [4,5]. Biochemical studies have demonstrated that Sic1 in its unphosphorylated form is immune from ubiquitination by SCFCdc4 and does not interact with Cdc4. However, phosphorylation of Sic1 complexed with inactive Clb5–Cdc28 by the Cln–Cdc28 kinase allows association of Sic1 with SCFCdc4 and its ubiquitination by Cdc34 [6,7,11]. We now know that this paradigm of phosphorylation-driven ubiquitination through an SCF complex is employed by numerous signaling pathways to control the stability of many regulatory proteins, including those involved in cell-cycle control (G1 cyclins, CDK inhibitors), transcription (β-catenin, IκBα, Gcn4), DNA replication (Cdc6) and others [4,5]. Linking multisite phosphorylation to an ultrasensitive cell-cycle transition
Although Sic1 is arguably the best understood ubiquitination substrate, aspects of its regulation remain murky. Of central importance is the question of how the Cln–Cdc28 threshold is set and how this information is imposed upon Sic1 to coordinate the timing of its destruction
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02238-3
TRENDS in Cell Biology Vol.12 No.3 March 2002
101
Research News
The Golgi apparatus: going round in circles? Francis A. Barr
Since the work of Palade and colleagues, protein transport from the endoplasmic reticulum (ER) and through the Golgi apparatus has been envisioned as a process primarily mediated by small vesicular carriers budding from one compartment and fusing with the next [1,2]. This view has been challenged in recent years by studies of the secretion of procollagen, a molecule simply too large to fit inside classical vesicular carriers of 60–70 nm diameter, which passes in an anterograde fashion across the Golgi stack without ever leaving the cisternae [3]. A cisternal maturation model was used to explain these data (Fig. 1). In such a model, Golgi cisternae arise by the fusion of vesicular or tubular intermediates from the ER and then mature from a cis- to a trans-form by the exchange of enzymes in small vesicular carriers moving in a retrograde fashion. Mironov and coworkers now extend their previous observations on procollagen to include the much smaller transmembrane glycoprotein of vesicular stomatitis virus (VSV-G) [4]. They find that neither cargo molecule enters transport vesicles while progressing through the Golgi stack, and these cargos are transported at the same rate, consistent with both being in the same maturing cisternal structure. Biochemical and morphological evidence supporting other aspects of the cisternal maturation model also exists, most importantly indicating that Golgi resident http://tcb.trends.com
PM
(a)
Golgi
TransMedial GTC
COP I
Cis-
COP I
rab1+p115 –
COP II
Sar1DN
ER
Recent studies have questioned the idea that the Golgi complex is a stable organelle with a unique identity through which secretory cargo is transported by vesicles. Instead, it is proposed that Golgi apparatus proteins continuously recycle via the endoplasmic reticulum by vesicle transport, whereas cargo molecules remain in maturing cisternal structures. Rather than forming a rigid matrix, structural Golgi proteins might be highly dynamic and recycle via the cytoplasm. I will discuss the evidence for these claims and consider whether or not they really disprove older ideas on how the Golgi apparatus is structured and performs its function.
(b)
Tethering complexes Enzymes Cargo TRENDS in Cell Biology
Fig. 1. A recycling model for Golgi traffic. (a) Golgi cisternae form by the fusion of transport intermediates arising from the COP II pathway. This can be blocked by dominant-negative forms of the Sar1 GTPase, which is required for the recruitment of COP II to endoplasmic reticulum (ER) membranes. The recognition and fusion of COP II vesicles involves the small GTPase rab1 and the tethering factor p115, plus a variety of other molecules needed to regulate rab1 activation [22]. Retrograde traffic between Golgi cisternae and back to the ER is mediated by COP I vesicles, and, within the Golgi, their tethering is likely to require the Golgi transport complex (GTC) [23,24] (b) Golgi enzymes recycle in COP I vesicles between adjacent cisternae and the ER. In this model, an important determinant of localization within the stack for resident proteins would be their rate of recycling in COP I vesicles – the lower the rate of recycling, the more trans the localization of the protein. These vesicles might also not be in free diffusion, being tethered by complexes such as the GTC or p115–GM130 from the moment of formation until reaching their destination.
proteins are sorted into recycling vesicles at the different levels of the Golgi stack [5–7]. Studies from the Nilsson lab describe the purification of different classes of COP I vesicles containing either cis- or medial-Golgi markers, but little anterograde cargo [6,8]. These vesicles select cargo by coupling the recruitment of
COP I complexes to GTP hydrolysis by the small GTPase ARF1. The mechanism for this appears to involve direct modulation of ARF1 GTPase-activating protein (GAP) activity by at least some transmembrane cargo molecules. The cytoplasmic domains of these proteins might inhibit the ARF-GAP activity,
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02240-1
Research Update
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TRENDS in Cell Biology Vol.12 No.3 March 2002
(a)
Resident proteins Matrix proteins
(b)
Tethering/matrix complexes TRENDS in Cell Biology
Fig. 2. The Golgi matrix. (a) Matrix proteins such as GM130 might form an ordered structure, giving long-range organization to Golgi cisternae. Interactions of this structure with resident proteins such as enzymes would be important for their localization within the stack. (b) Alternatively, matrix proteins might function in much smaller units linking adjacent cisternae and serving to give compartmental identity during membrane traffic as part of tethering complexes. It is quite possible that both these proposals are correct, and different Golgi matrix proteins function in one or both of these ways.
allowing accumulation of active ARF1 on the membrane, thus promoting COP I recruitment and vesicle formation [8,9]. These findings that some, if not all, anterograde cargo is transported in maturing cisternae, while enzymes are present in COP I vesicles, support the maturation model for Golgi transport and therefore also raise issues concerning the maintenance of the stacked cisternal structure of the Golgi apparatus. A maturation model suggests that structural protein complexes, often referred to as the ‘Golgi matrix’, would also need to be highly dynamic, recycling as the cisternae mature, whereas previously they were considered to be relatively static components of long-lived cisternal structures. The Golgi matrix: is it dynamic or static?
The putative Golgi matrix was originally described biochemically and morphologically as a proteinaceous structure isolated from Golgi membranes that retained the form of Golgi cisternae http://tcb.trends.com
and was capable of specifically binding to Golgi enzymes (Fig. 2) [10]. Purified Golgi membranes retain their characteristic stacked cisternal structure, and it was proposed that the Golgi matrix proteins formed an exoskeleton required to maintain this unique organization. Many components of this Golgi matrix are coiled-coil proteins, the best characterized of which are the cis-Golgi proteins GM130 and its interacting partner p115 [11,12]. GM130 is targeted to Golgi membranes through an interaction with GRASP65, a protein identified in an in vitro functional assay for factors required for the stacking of Golgi cisternae [13]. It is interesting to note that GRASP65 is a myristoylated protein and that many other proteins with this modification can switch between a membrane-bound and cytosolic form in response to a trigger such as GTP binding [13,14]. A recent study on the dynamics of a GFP-tagged form of GRASP65 in vivo found that it appears to rapidly recycle on and off Golgi membranes [15]. Whether or not it remains bound to GM130 in these
experiments is unclear, but previous biochemical studies have found that GM130 and GRASP65 interact with high affinity, making it likely that the complex as a whole is recycling [13]. This study also found that there was a cytosolic pool of GRASP65 – despite the fact that this was not observed in previous work on the GRASP proteins. Another recent paper investigating how cis-Golgi matrix proteins target to the Golgi showed that newly synthesized endogenous GRASP65 and GM130 bind rapidly and directly to the Golgi, and in these experiments there was no evidence for a cytosolic pool of the two proteins [16]. This obviously presents a disparity that is impossible to resolve at the present time and will require further investigation. How can these findings be explained? One possibility is that the GRASP65–GM130 complex actively recycles from Golgi membranes to the cytosol, there being only a very small pool in the cytosol at any one time. Understanding the mechanism of matrix protein targeting to the highly dynamic Golgi cisternae and how this is regulated will be important for resolving this issue. Finally, it should also be remembered that exchange of membrane-associated Golgi matrix subunits with a soluble pool does not in itself prove that the cisterna is changing its identity. Does the Golgi matrix exist?
It is often stated that ‘seeing is believing’, and, because a Golgi matrix has been visualized by microscopy, it might seem odd to question the existence of such a structure. As originally envisioned, this structure should comprise a stable assembly of structural proteins imparting organization and identity to Golgi membranes. Warren and coworkers, who found that a Golgi-like structure exists independently of protein transport from the endoplasmic reticulum, provided evidence for this rigid view of the Golgi matrix [10,17]. In their experiments, these matrix structures resembled a Golgi apparatus under the light microscope in vivo, but unfortunately, under the electron microscope, they lacked a clear cisternal organization [17]. Obviously, more than just a Golgi matrix is required to build a Golgi apparatus, although these data do support the idea of a Golgi matrix structure independent of the ER. Two other studies have re-examined this evidence using microinjection or
Research Update
overexpression of dominant-negative Sar1 mutants to block ER exit by the COP-II vesicle transport pathway [15,18]. In both cases, Golgi proteins redistributed to the ER, and the Golgi apparatus disappeared. The kinetics of relocalization were different for Golgi enzymes compared with structural Golgi matrix proteins; the latter redistribute more slowly, and this effect requires higher concentrations of dominant-negative Sar1 [18]. While these observations do not really argue against a Golgi matrix imparting structure to the Golgi apparatus, they do question the independence of such a structure from the ER. However, a caveat to this is that it does not prove that Golgi matrix proteins ever recycle back to the ER under normal conditions. A second issue is that it takes many hours for Golgi matrix proteins to redistribute to the ER in these experiments, and, at shorter times, no effect on Golgi matrix proteins such as GM130 and GRASP65 is observed under comparable conditions [16]. However, even this point is disputed as other studies find a rapid redistribution of Golgi matrix proteins in minutes under apparently similar conditions [15]. Rather than debate the existence of such a matrix, it is probably more useful to ask whether its potential components are important for maintaining the Golgi and what their functions might be. GM130 is known to interact both with Golgi Rab proteins and with the vesicle tethering factor p115 and to play a role in the formation of Golgi stacks in a cell-free assay [19,20]. A novel Golgi matrix protein, golgin-45 was recently described as the binding partner for GRASP55 on the medial-Golgi [21]. Although the function of golgin-45 is not known, it binds to the small GTPase rab2, implicating it in some aspect of vesicle tethering to the medial-Golgi. Depletion of golgin-45 by RNA interference causes Golgi enzymes to collapse back into the ER, and other Golgi matrix proteins to redistribute to small punctate structures and the cytoplasm [21]. Not surprisingly this also resulted in a block to protein transport as there was no longer a functional Golgi apparatus. In summary, although the Golgi matrix proteins do seem to be important for maintenance of Golgi structure and to impart Golgi identity, the existence of an ordered structure justifying the term matrix is less clear. Rather than a static matrix, an alternative model should be http://tcb.trends.com
TRENDS in Cell Biology Vol.12 No.3 March 2002
103
considered in which these proteins exist in smaller landmark complexes specific for the different Golgi cisternae such as GRASP65–GM130–p115 on the cis-Golgi, and GRASP55–golgin-45 on the medial-Golgi to give compartmental identity (Fig. 2).
material from the ER to the plasma membrane? Currently, that there is a large recycling flux of resident proteins is the most popular explanation, but, as with many aspects of the Golgi, this is a circular argument subject to change. Acknowledgements
Concluding remarks
Because the secretory pathway is primarily a cargo transport system, it makes sense that small quantities of enzymes are taken to meet the large amount of substrates they need to modify. This would seem to be more effective than the converse situation, transporting large amounts of cargo in small vesicular structures. However, there are still some unresolved issues with this model. It has been reported that Golgi enzymes recycle via the ER and that all forward-moving cargo, presumably including enzymes, moves at the same rate through the Golgi apparatus. If this were the case, then the cis-Golgi would contain not only cis-enzymes but also significant amounts of recycled medial- and trans-enzymes on their way back to their correct locations. How then can their discrete distributions to the different layers of the Golgi be explained? The most parsimonious explanation is that a sequential enzyme recycling mechanism exists that transports enzymes to the preceding cisterna in vesicles, rather than allowing them to go directly back to the ER. If these vesicles were not in free diffusion but tethered to the adjacent destination compartment from the moment of formation, this could explain such a mechanism. To some extent, this is merely postponing the problem for a later date as the molecular details of cis-Golgi versus medial- or trans-Golgi localization for particular proteins still have to be explained. A second issue relates to the putative Golgi matrix proteins and how they are specifically targeted and then retain their localization to different cisternae within the stack. As discussed above, it is possible that some matrix proteins recycle via the cytoplasm, but others are integral membrane proteins and must therefore recycle in vesicles. Despite the shift in perspective from considering the Golgi apparatus as a static organelle to a more dynamic structure, the essential cell-biological question remains the same: how is this structure maintained in the face of a large flow of secretory
I thank Ben Short and Sean Munro for discussions during the course of writing this article and acknowledge the comments of the reviewers. References 1 Farquhar, M.G. and Palade, G.E. (1998) The Golgi apparatus – 100 years of progress and controversy. Trends Cell Biol. 8, 2–10 2 Rothman, J.E. and Orci, L. (1992) Molecular dissection of the secretory pathway. Nature 355, 409–415 3 Bonfanti, L. et al. (1998) Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation. Cell 95, 993–1003 4 Mironov, A.A. et al. (2001) Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the lumen of cisternae. J. Cell Biol. 155, 1225–1238 5 Love, H.D. et al. (1998) Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport. J. Cell Biol. 140, 541–551 6 Lanoix, J. et al. (1999) GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles. EMBO J. 18, 4935–4948 7 Martínez-Menárguez, J.A. et al. (2001) Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisterna-progression model of intra-Golgi transport. J. Cell Biol. 155, 1213–1224 8 Lanoix, J. et al. (2001) Sorting of Golgi resident proteins into different sub populations of COPI vesicles: A role for ArfGAP1. J. Cell Biol. 155, 1199–1212 9 Goldberg, J. (2000) Decoding of sorting signals by coatomer through a GTPase switch in the COPI coat complex. Cell 100, 671–679 10 Slusarewicz, P. et al. (1994) Isolation of a matrix that binds medial Golgi enzymes. J. Cell Biol. 124, 405–413 11 Fritzler, M.J. et al. (1993) Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex. J. Exp. Med. 178, 49–62 12 Nakamura, N. et al. (1997) The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner. Cell 89, 445–455 13 Barr, F.A. et al. (1997) GRASP65, a protein involved in the stacking of Golgi cisternae. Cell 91, 253–262 14 Goldberg, J. (1998) Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95, 237–248 15 Ward, T.H. et al. (2001) Maintenance of Golgi structure and function depends on the integrity of ER export. J. Cell Biol. 155, 557–570
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cisternae in a cell-free system. J. Cell Biol. 146, 57–70 Weide, T. et al. (2001) The Golgi matrix protein GM130: a specific interacting partner of the small GTPase rab1b. EMBO Rep. 2, 336–341 Short, B. et al. (2001) A GRASP55–rab2 effector complex linking Golgi structure to membrane traffic. J. Cell Biol. 155, 877–884 Allan, B.B. et al. (2000) Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Science 289, 444–448 Whyte, J.R.C. and Munro, S. (2001) The Sec34/35 Golgi transport complex is related to the exocyst,
defining a family of complexes involved in multiple steps of membrane traffic. Dev. Cell 1, 527–537 24 Walter, D.M. et al. (1998) Purification and characterization of a novel 13 S hetero-oligomeric protein complex that stimulates in vitro Golgi transport. J. Biol. Chem. 273, 29565–29576
Francis A. Barr Dept of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, Martinsried, 82152 Germany. e-mail: [email protected]
A phosphorylation-driven ubiquitination switch for cell-cycle control J. Wade Harper Cellular changes in state can be dictated by complex all-or-nothing switches built from ultrasensitive protein kinase cascades, positive-feedback loops and other mechanisms. Recent work has established that phosphorylation-driven protein destruction through the SCF ubiquitinligase pathway can also occur in a switchlike manner. In this context, multiple phosphorylation events are used to set a threshold for substrate targeting, thereby providing a framework for understanding the inter-relationship between protein phosphorylation and ubiquitin-mediated proteolysis.
The ability of complex cellular systems to undergo coordinate and irreversible changes in state is fundamental to the proper development of an organism. Such transitions frequently involve one or more molecular switches, capable of coordinately converting an entire pool of a crucial regulatory factor from one activity state to another over an extremely small time interval. Molecular switches often involve protein kinase cascades in which the response of the terminal kinase is ultrasensitive to the level of a continuously variable stimulus and is re-enforced by positive-feedback mechanisms [1]. In essence, the molecular circuitry inherent in an ultrasensitive pathway determines the threshold stimulus required for turning on the pathway in an all-or-nothing manner. Now new work [2] has established that phosphorylation-driven ubiquitin-mediated destruction of the cyclin-dependent kinase (CDK) inhibitor Sic1 may also occur in a http://tcb.trends.com
switch-like fashion during cell-cycle progression and, in so doing, sets the threshold for the level of upstream kinase activity required to turn on the destruction switch and activate DNA synthesis. In order to duplicate itself, a cell must make a copy of its genome through the process of DNA replication (S phase) and then segregate two equal copies of this genetic material into mother and daughter cells through the process of mitosis. The decision to enter into a new round of cell division is not taken lightly, and multiple control mechanisms are used to control the decision to move from the G1 phase of the cell cycle into the replicative stage. Entry into S phase in the budding yeast Saccharomyces cerevisiae requires Clb5–Cdc28 kinase, which is kept inactive during the G1 phase by the CDK inhibitor Sic1 [3]. During this period, cells sense environmental nutrients and convert these signals into G1 cyclin (Cln)–Cdc28 kinase activity (Fig. 1a). Once threshold levels of Cln–Cdc28 activity are achieved, Sic1 is rapidly phosphorylated, ubiquitinated and destroyed, thereby releasing Clb5–Cdc28 to activate S phase [4,5]. The necessity of properly controlling Sic1 destruction is revealed by the fact that cells lacking SIC1 begin DNA synthesis prematurely and display genomic instability. Sic1 ubiquitination occurs through the concerted action of E1 ubiquitinactivating enzyme, the E2 conjugating enzyme Cdc34 and an E3 ubiquitin ligase referred to as SCFCdc4 [6,7]. This ubiquitin ligase comprises Skp1, Cdc53/Cul1, Rbx1
(also called Hrt1 or Roc1) and Cdc4. Temperature-sensitive mutations in each subunit of SCFCdc4 cause G1 arrest and Sic1 stabilization in budding yeast. Skp1–Cdc53–Rbx1 forms a core ubiquitin ligase that interacts with E2s [8,9], while Cdc4, a member of the F-box family of proteins10, interacts with Skp1 through the F-box motif and with substrates through C-terminal WD40 repeats [7,10]. F-box proteins are a large family of proteins that generally serve as receptors for ubiquitination substrates [4,5]. Biochemical studies have demonstrated that Sic1 in its unphosphorylated form is immune from ubiquitination by SCFCdc4 and does not interact with Cdc4. However, phosphorylation of Sic1 complexed with inactive Clb5–Cdc28 by the Cln–Cdc28 kinase allows association of Sic1 with SCFCdc4 and its ubiquitination by Cdc34 [6,7,11]. We now know that this paradigm of phosphorylation-driven ubiquitination through an SCF complex is employed by numerous signaling pathways to control the stability of many regulatory proteins, including those involved in cell-cycle control (G1 cyclins, CDK inhibitors), transcription (β-catenin, IκBα, Gcn4), DNA replication (Cdc6) and others [4,5]. Linking multisite phosphorylation to an ultrasensitive cell-cycle transition
Although Sic1 is arguably the best understood ubiquitination substrate, aspects of its regulation remain murky. Of central importance is the question of how the Cln–Cdc28 threshold is set and how this information is imposed upon Sic1 to coordinate the timing of its destruction
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