SCIENTIFIC REVIEW Kinetics and Energetics of Protein Sorting

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1 Kinetics and Energetics of Protein Sorting Charlotte Grove Department of Neuroscience Albert Einstein College of Medicine Bronx, New York ABSTRACT Currently, there are four models for the sorting of protein into different cellular compartments. Each model makes an attempt to explain how the interactions of the essential components of vesicle formation, ARF1 or Sar1 GTPase, GTPase activating protein, coatomer proteins, and p24 proteins, lead to high fidelity protein sorting. Each model is supported by a subset of the available genetic, morphological, biochemical, and kinetic data. INTRODUCTION Proteins expressed in the cell undergo regulated folding in the endoplasmic reticulum (ER) and must be delivered to their appropriate cellular compartments. Sorting occurs in the ER and Golgi apparatus. The early stages of protein sorting ensure that only the appropriate, highly concentrated and properly folded proteins are transported. A protein destined for the plasma membrane travels in an anterograde direction from the ER to the cis-golgi then to the medial-golgi and finally to the trans-golgi before it is transported to the cell surface. Retrograde transport is necessary for resident proteins that have escaped their ER or specific Golgi network domain or that shuttle between several cell domains to fulfill their cellular role (e.g., cargo receptors). Proteins that remain in the ER (ER resident proteins) do not contain the diphenylalanine (FF) motif (Sohn et al., 1996) and/or diarginine (RR) motif (Goldberg, 2000). These motifs, FF and RR, are present on the cytosolic tails of some p24 cargo receptor proteins and target them for a destination downstream from the ER. On the other hand a dilysine (KK) motif is expressed on ER resident transmembrane proteins that undergo retrieval via retrograde transport from the Golgi to the ER. Vesicles are the transport containers for the selected proteins. The shells surrounding vesicles moving in an anterograde fashionconsist of the four subunit coatomer protein complex II (COPII) whereas the shells of vesicles moving in a retrograde or bi-directional fashion (Goldberg, 2000; Szafer et al., 2001) consist of the seven subunit coatomer protein complex I (COPI). Protein sorting appears to be thermodynamically enigmatic. Certainly it has to take place at one or many places along the secretory pathway to allow for protein concentration required by the cell to carry out its many functions. Nevertheless, the concentration of proteins against a gradient or from an energetic standpoint, a decrease in entropy, breaks the second law of thermodynamics, unless a compensating enthalpic energy term can be identified. If proteins are able to form oligomeric complexes within the ER and other organelles, the formation of bonds in such complexes, might be able to compensate for the reduction in entropy. Another possible concentrating mechanism would be the selection of proteins based on kinetics. Certain molecular associations might exist within a window of time and be stable long enough for vesicles to be pinched off thereby trapping a concentrated form of several proteins (Goldberg, 2000). Prior to a discussion of the models that arise from consideration of the oligomeric complex formation and kinetic sorting themes, current findings of the key components in vesicle formation and their interactions will be reviewed. REQUIREMENTS FOR VESICLE BUD FORMATION Several key components are required for vesicle bud formation in vivo (Figure 1). These include a GTPase or GTP binding protein, an enzyme that binds and hydrolyzes GTP as well as attaches to the cytoplasmic side of the ER or Golgi apparatus, a GTPase activating factor (GAP), a coat complex that polymerizes to form the vesicle shell (COPI or COPII), and a p24 transmembrane proteins that, as putative cargo receptors, ensure protein sorting fidelity as well as select the cargo proteins that are encased in the vesicle. Small GTP binding proteins play a significant role in the secretory pathway. GTP hydrolysis is coupled with and regulates both protein sorting and transport direction. GTPases (ARF1 for COPI and Sar1 for COPII) are soluble cytosolic proteins in the GDP bound form and can become associated with a donor membrane anchored by a myristoylated tail in the GTP bound form (Eugster et. al., 2000). A guanine nucleotide exchange factor (GEF) converts the GTPase into the GTP bound form (Figure 1a) (Zhao et al., 1999; Matsuoka et al., 1998; Sprang et al., 1998). Upon binding of a GTPase to the donor membrane, a coatomer complex, COPI or COPII complex, is recruited to the membrane. The COPI complex contains dual binding sites, a site for ARF1 interaction on β-copi subunit and a site for transmembrane proteins such as p24 family members, ERGIC53, and v-snares (Bremser et al., 1999; Springer and Scheckman, 1998; Dominguez et al.,1998), on the γ-copi subunit (Figure 1c) (Zhao et al., 1999; Harter et al., 1998). Subsequent to the interaction of these COPI subunits with ARF1 and the p24 transmembrane proteins, the COPs polymerize and initiate vesicle shell formation (Figure 1d and Figure 1e). Einstein Quart. J. Biol. Med. (2002) 19:

2 Although it has been demonstrated that bud formation can take place in vitro without the presence of p24 components (Bremser et al., 1999; Yeung et al., 1995; Springer et al., 2000), in vivo these transmembrane proteins are found in all forms of vesicles: anterograde Sar1/COPII and retrograde ARF1/COPI (Stammes et al., 1995; Schmmoller et al., 1995; Sohn et al., 1996). p24 transmembrane proteins may have the role of enhancing the protein sorting fidelity (Kaiser, 2000). In vivo the interaction of p24 cytosolic tails with the γ-copi subunit may induce a conformational change in the COPI complex that enables polymerization of COPI and subsequent vesicle formation (Zhao et al., 1999). Because of their ubiquitous nature, p24 proteins provide a useful organizing theme from which to consider the various models of protein sorting (Kaiser, 2000; Goldberg, 2000). Additionally, the p24 proteins figure prominently in both the kinetic and thermodynamic models of protein sorting. p24 TRANSMEMBRANE PROTEIN STRUCTURE AND SEQUENCE There are eight members of the p24 transmembrane protein family (Table 1) (Schimmoller et al., 1995; Stamnes et al., 1995; Belden and Borlow, 1996; Blum et al., 1996; Sohn et al., 1996; Dominguez et al., 1998; Nakamura et al.,1998; Lavoie et al., 1999; Marzioch et al.; 1999; Springer, 2000). They are type I integral membrane proteins with short cytoplasmic tails and larger lumenal domains. This family of proteins is subdivided based on their degree of homology to each other (Dominguez et al., 1998) with each subfamily given a letter (α, β, δ, and γ). Members within a subfamily are distinguished by a number, which indicates their order of discovery (Fullerkrug et al., 1999). Additionally, letters preceding 24 indicate the species from which the protein is derived. p24 proteins refers to the entire family and not just the hp24β1 subfamily, which has also been termed p24 in the literature. The FF motif on the cytosolic tails of the p24 proteins enables them to bind to COPII for anterograde transport, and the K(X)KXX acts a retrieval motif for retrograde transport. The longer F/YXXXXF/Y motif in the cytoplasmic tails of the p24 proteins may enable them to bind GTPase activating protein for uptake into a vesicle (Dominguez et al., 1998; Lanoix et al., 2001). Site directed photocrosslinking is used to elucidate the interactions of ARF1 and p24 proteins with COPI. With this technique one endogenous amino acid residue interacting with a residue of another protein is replaced by a photoliable amino acid analog. Ultraviolet excitation of the photoactive moiety gives rise to a reactive free radical state that enables the substituted residue to covalently crosslink with other amino acid residues in very close proximity. The subsequent, protein-protein interaction, reflected in crossslinked product, can be isolated and analyzed by immunoprecipitation (Fischer et al., 2000). In site directed photocrosslinking experiments, the p24 protein tails have been shown to interact with the γ-copi subunit (Zhao et al., 1999). The γ-copi subunit is able to bind the dilysine ER retrieval motif as TABLE I Members of the p24 transmenbrane protein family Protein Alternate Names Consensus Sequences Comments hp24α1 gp251, Erp1p, Erp6p FF K(X)KXX calnexin-binding protein of the ER FF hp24α2 GMP25, Erp5p K(X)KXX coupled with COPI leads to vesicular CQMRHLKSFFEAKKLV tubular clusters (VTC) formation hp24β1 hp24δ1 hp24γ1 hp24γ2 emp24, p24 erv25, p23 T1/ST2, Erp2, Erp3, Erp4 T1/ST2150 R(X)RXX CQIYYLKRFFEVRRVV K(X)KXX CQVFYLRRFFKAKKLIE hp24γ3 gp27 CQVFLLKSFFSDKRTTTTRVGS hp24γ4 p26 CQVLLLKSFFTEKRPISRAVHS 114 EINSTEIN QUARTERLY, Copyright 2002

3 FIGURE 1 This schematic combines aspects of the kinetic models proposed by Lanoix et al.(2001) and Goldberg (2000) and illustrates the proposed interactions between the key components involved in vesicle formation. These include a GEF, GTPase, GAP, COP protein complex that forms the shell around the vesicle, p24 proteins, and cargo proteins. a) Souble GDP bound ARF1 exchanges GDP for GTP through interaction with GEF and becomes associated with the membrane in its myristoylated form. b) Non-p24 protein tails are sampled by ARF1, GAP, and COP. Hydrolysis is not inhibited and the non-p24 protein is deselected. c) p24 protein tails are sampled by ARF1, GAP, and COP. Hydrolysis is inhibited and the p24 protein(s) are stabilized in a priming complex, ready for coatomer polymerization. d) The stable priming complexes undergo polymerization via coatomer-coatomer interactions and the vesicle bud begins to form. e) A three dimensional view of priming complex polymerization suggests some of the mechanical forces that the priming complexes and in particular the coatomer proteins probably have to accommodate (adapted from Goldberg, 2000; Lanoix et al., 2001; Springer, 1999; Zhao et al.,1999). The Einstein Quarterly Journal of Biology and Medicine 115

4 well as the diphenylalanine COP binding motif (Fiedler et al., 1996; Sohn et al., 1996). In fact, proteins with these motifs compete for the same site on the γ-copi subunit (Harter et al., 1996; Harteraud Wieland 1998; Goldberg, 2000). Site directed photocrosslinking experiments also indicate that the effector loop of ARF1 interacts with the coatomer complex at the β- and γ-copi subunit interface. These results imply that a bivalent interaction of the COPI complex with ARF1 and with p24 proteins is part of bud formation (Zhao et al., 1999). A number of analyses indicate that the p24 proteins can form oligomeric complexes (Springer et al., 2000; Lavoie et al., 1999; Fullekrug et al., 1999; Marzioch et al., 1999). It is not yet clear how the cytoplasmic tails of such a complex would interact with the COPI coat as it is depicted in Figure 1c (Zhao et al., 1999). Several tails are depicted as interacting with the γ-copi subunit site. The interaction of these p24 proteins with COP may induce a conformational change in γ-copi, which promotes polymerization of the comatomer complex (Rheinhard et al., 1999). The α2, β1, δ1, and γ3 p24 proteins must form a hetero-oligomeric complex to leave the ER in COPII vesicles and may have to form an analogous complex in order to be taken up into COPI vesicles. MODELS OF PROTEIN SORTING The models for protein sorting by vesicles can be characterized on the basis of whether the COP-p24 protein interactions are stabilized thermodynamically or kinetically. All models, kinetic and thermodynamic, depend on polymerization of the COP subunits to capture selected transmembrane cargo and/or cargo receptor proteins. One model (Figure 1a-e) is kinetic, while three other models (Figure 2a-c) are thermodynamic. In the kinetic model, the p24 proteins concentrate to form a subdomain in the membrane only if the interaction between the p24 protein tail and COP is transiently stabilized by the inhibition of ARF1/COP hydrolysis (Figure 1c). If hydrolysis occurs before coatomer polymerization occurs, as would be the case with a non-p24 protein tail, the protein is discarded from the nascent vesicle (Figure 1b). By contrast, in the three other models the selection of vesicle cargo depends on a thermodynamically stable interaction between vesicle components (Schmid, 1997; Springer and Scheckman, 1998; Springer et al., 1999; Bremser et al., 1999; Haucke and DeCamilli, 1999; Kirchausen et al., 1997). Either p24 proteins with their associated cargo become concentrated, because they form stable associations with COPs that polymerize or because stable p24 protein-p24 protein associations form rafts in the membrane that form stable complexes with COPs. These represent thermodynamic models, whereby the unfavorable reduction of entropy, the concentration of p24 proteins, is offset by a favorable increase in enthalpy, the stabilizing interactions between proteins. KINETIC MODELS Goldberg (2000) and Lanoix et al. (2001) put forth the kinetic argument that at least initially, the regulation of the coat assembly is controlled by GTP hydrolysis. This hydrolysis or lack of hydrolysis is a function of a concerted interaction between ARF1, COP subuints β and γ, sequences on the cytoplasmic tails of the p24 proteins and GAP. Zhao et al. (1999) and Goldberg (2000), respectively, have demonstrated that the binding sites on COPs for p24 proteins and ARF1 GTPase are located in a confined area and are allosterically related to one another. COP-dependent GTP hydrolysis can be thought of as an enhancement of GAP dependent GTP hydrolysis. As mentioned, in the initial budding process, the p24 proteins that interact with coat proteins and/or GAP and at the same time inhibit GTP hydrolysis will have a greater probability of moving to the later stages of the budding process. In the late stage, the effect of GTP hydrolysis (dissociation of p24/coat protein) may be less significant, since some degree of coat polymerization (Reinhard et al., 1999) and cargo-cargo interaction located in the lumen of the vesicle bud may have already stabilized the complex (Zhu et al., 1998; Springer et al, 1999). If the kinetics of GTP hydrolysis do indeed provide a protein selection function, GTP hydrolysis must occur primarily only in the presence of bound COPs (Goldberg, 2000). Lanoix et al. (2001) like Goldberg (2000) agrees that inhibition of GTP hydrolysis is critical for the uptake of cargo into vesicles. In contrast to Goldberg s model, he proposes a two-step process that requires GTP hydrolysis for deselection of non-p24 protein and non-cargo proteins, but subsequently requires the inhibition of hydrolysis for vesicle formation that incorporates p24 and cargo proteins. Additionally, in the Lanoix et al. (2001) model this inhibition is not a function of a p24 protein-cop interaction, but rather that of a p24 protein-gap interaction. ARF1-GAP Interaction ARF1, a member of the Ras GTPase superfamily, appears to be unique among the Ras GTPases in that the tripartite complex of ARF1-GTP, ARF1GAP, and coat protein (Springer et al., 1999; Bremser et al., 1999; Matsukoka et al., 1998; Sprang, 1998) or its analog (Sar1 complex) is required for rapid GTP hydrolysis (Goldberg, 1999; Yoshihisa et al., 1993). In general, GAP interaction with GTPases orients and stabilizes the GTPase residues of the GTP hydrolysis site in an active conformation. An arginine residue, supplied by the GAP in the case of Ras and Rho GTPases, binds to the terminal phosphate group of the GTP, thereby stabilizing the transition state (Rittinger et al., 1997). Such an arginine residue or finger is absent in both ARF1 and ARF1GAP. Goldberg (1999) showed that COP-dependent hydrolysis enhances the rate of ARF1- GTP hydrolysis by a factor of 1000 due to ARF1GAP dependent hydrolysis. The COP may supply an arginine 116 EINSTEIN QUARTERLY, Copyright 2002

5 FIGURE 2 a) Cargo exclusion Cargo proteins that bind with high affinity to the interior surface of the vesicle may be able to displace p24 proteins, which act as placeholders in the nascent vesicle. ER resident proteins are unable to displace p24 proteins and are excluded from the vesicle. b) Time delay The presence of p24 proteins may slow the polymerization of the vesicle coat, thereby enabling the sorting process to reach completion. c) Membrane segregation Specialized membrane domains form when p24 proteins self associate. The p24 protein membrane domain may serve as a substrate for vesicle coat formation as well as for cargo protein selection (adapted from Kaiser, 2000). finger for insertion into the ARF1 catalytic site. The correct conformation to enable insertion of the arginine finger, and subsequent efficient hydrolysis, may be induced by the tripartite interaction of ARF1, ARF1GAP, and COP. However, this role for COP has been challenged when ARF1 of the tripartite complex is in its full-length myristoylated form, unlike the mutated form used by Goldberg (1999) and Szafer et al. (2000). The active hydrolysis conformation may be modified and made less optimal for hydrolysis by the interaction of the p24 protein cytosolic tails with ARF1GAP (Lanoix et al., 2001), rather than by the interaction of p24 protein cytosolic tails with γ-copi subunit of the COP complex (Goldberg, 2000). COP-ARF1 Interaction A GAP is required for GTP hydrolysis to occur. However, COP interaction with ARF1 in addition to ARF1GAP interaction can significantly increase the rate of hydrolysis (Goldberg, 1999; Szafer et al., 2001). By using the appropriate ratio of ARF1:COP, Goldberg (1999) was able to demonstrate that the rate of COP dependent GTP hydrolysis could be influenced by the motif on the p24 protein cytosolic tail. With a 1:6.5 COP to ARF1 ratio, the rate of the GAP dependent hydrolysis was reduced to 1/100 that of rate due to the influence of COP. Although this ratio is considered to be higher than that in physiologic circum- The Einstein Quarterly Journal of Biology and Medicine 117

6 stances, it afforded a sensitive measurement of the p24 protein tail influence on COP-dependent hydrolysis. The rate of COP-dependent hydrolysis modulated by a high dose of bound p24β1 protein signal domain, that is the cytoplasmic tail containing a R(X)RXX motif, was reduced by a factor of 50 relative to the control. In contrast, a signal domain having an retrieval motif, K(X)KXX had no influence on the rate of COP-dependent hydrolysis. The p24β1 signal domain may turn off COP based GTP hydrolysis by changing the position and thus the function of the catalytic residues supplied by COPI to the ARF1 GTPase active site. This difference in COP-dependent hydrolysis, coupled with competition of these proteins (containing p24 and ER resident protein motifs) for the same binding site on the COP (Harter et al.,1998; Zhao et al, 1999; Goldberg, 2000) are the ingredients necessary for a protein sorting system. In this system, the COP has a differential response to various cytoplasmic tail motifs. According to Goldberg (2000), although GAP activity is necessary for GTP hydroysis, this activity by itself is insufficient to provide the differential response that the COP GTP hydrolysis activity provides. As noted previously, Lanoix et al. (2001) proposes that the GTP hydrolysis modulated by a p24 protein-gap interaction does provide this differential response. The F/YXXXXF/Y motif in the p24 protein tails may enable binding to GAP for uptake into a vesicle (Dominguez et al., 1998; Lanoix et al., 2001). THERMODYNAMIC MODELS The difficulty in accruing structural evidence for oligomeric complexes, having at least two-dimensional short-range order, is that the analytic tools available lack the necessary spatial and temporal resolution (Kaiser, 2000). For example diffraction techniques often employed are not able to detect a sufficiently strong signal (either through probing a large enough quantity of the complex or signal averaging over an extended time) to detect the tiny amounts of proposed transient complexes. Nevertheless, some evidence for oligomeric complexes and kinetic sorting has emerged in the COPI and COPII systems (Springer et al., 2000; Lavoie et al., 1999; Fullekrug et al., 1999; Marzioch et al. 1999). The model of protein sorting from the ER must provide a means (Kaiser, 2000) to explain the reduction in sorting efficiency exhibited when various members of the p24 family are mutated or deleted (Elrod-Erickson and Kaiser, 1996). The three current models, (Figure 2a-c) (Kaiser, 2000) all require a concentration of p24 proteins and/or COP as a precursor to high fidelity protein sorting. Here in lies a weakness of these models. By what mechanism(s) are particular p24 proteins and COPs concentrated? Presently there is limited evidence for the existence of subdomains in ER or Golgi apparatus membranes containing high concentrations of specific p24 proteins independent of a stabilizing COP. This suggests that the COP might supply a selection mechanism. If this were the case, particular COP subunits able to uniquely bind specific p24 proteins or perhaps a subset of the eight p24 proteins listed earlier, would somehow have to become concentrated within the COP polymer. Alternatively, if only one COP subunit interacted with p24 proteins (Zhao et al.,1999) and this subunit had only one isoform, the specific p24 proteins or a subset of p24 proteins would have to form oligiomeric complexes to ensure that COP binding sites became locally populated with a particular p24 protein or a p24 protein family subset. Lavoie et al. (1999) has provided some evidence of p24 protein concentrations contributing to the formation of vesicular tubular clusters (VTC), an ER subdomain thought to be the center of ER sorting in mammals. Nevertheless, structural corroboration for oligomeric complexes inside the vesicle remains difficult to obtain, as pointed out earlier, given the limitations of diffraction techniques. In the cargo exclusion model (Figure 2a), the spacing of p24 proteins, a function of their abundant nature and subsequent interaction with the COP, results in dense packing of the p24 proteins on the lumenal side of the vesicle. If a cargo protein has a high affinity for the interior surface of the bud, it is speculated that the cargo protein would replace the p24 protein, which had been acting as a placeholder. Given that the p24 proteins form a layer in contact with the COP via their cytosolic tails, by what mechanism would the soluble cargo protein interact/communicate with the COP before it replaces the p24 protein? Cargo molecules would be accepted or excluded based on their size relative to the spacing of the p24 proteins. If the p24 protein lumenal heads are quite large, only relatively small cargo molecules could be accommodated. Assuming that the observed combinations of p24 protein types accomplish sorting in this manner, one would expect to see cargo sorted according to size. This has yet to be examined. Conceivably, cargo protein size would be determined by the number, location, and affinity of remaining p24 proteins present (after the removal of the placeholder proteins) for interaction with the cargo protein. This interaction of the lumenal portion of the remaining p24 proteins with the cargo protein would stabilize the cargo protein proximal to the membrane primed for uptake in the nascent vesicle. The time delay model (Figure 2b) assumes that if COP polymerization happens too rapidly, cargo as well as ER resident molecules will not be able to find their appropriate receptors and the transfer rate of ER resident proteins to the Golgi apparatus would increase while that of non-resident ER proteins would decrease. In this model, the p24 proteins slow the formation of the vesicle, enabling cargo proteins to find their appropriate binding sites, while ER resident proteins diffuse away from the vesicle bud. Genetic evidence indicates that at least a subset of p24 proteins fulfill this role (Elrod-Erickson and Kaiser, 1996). Crosslink experiments have not yet successfully identified a direct connection between p24 and cargo proteins (Kaiser, 2000) and suggest that the link 118 EINSTEIN QUARTERLY, Copyright 2002

7 might be indirect. If the formation of an adapter layer of proteins between p24 and cargo proteins is indeed necessary, the time delay role of p24 proteins proposed in this model would be crucial. Specificity in cargo selection might be provided by the combination of particular p24 and adaptor proteins. In the membrane segregation model (Figure 2c), consistent with biochemical data (Marzioch et al., 1999; Fullekrug et al., 1999) that indicates several p24 protein types associate with each other. p24 proteins may form a raft in the ER membrane (Dominguez et al., 1998). These rafts might serve as recruitment sites for both cargo molecules in the ER or Golgi apparatus lumen and COP molecules in the cytosol. This model is consistent with the formation of VTCs (Lavoie et al., 1999). Nevertheless, to fully understand the mechanism of high fidelity sorting, the details of cargo and p24 proteins interactions have to be clarified for all of these models. CONCLUSION The cell accomplishes the heroic task of sorting a huge number of diverse proteins with efficiency and high fidelity. Additionally, it transports these proteins to varied locations throughout the cell in a timely fashion and in the correct amounts. Much has yet to be learned about the mechanisms involved in this task. Ideally techniques with sufficient spatial and temporal resolution would provide a structural understanding of the extent of p24 and cargo protein oligomerization inside vesicles. This would certainly help distinguish between the likelihood of the kinetic model and the three thermodynamic models, namely cargo exclusion, time delay and membrane segregation models presented (Kaiser, 2000). However, the availability of such techniques seems remote. For the near term, advances may come from in vitro dissection of vesicle formation and improvements in imaging techniques of live cells (Kaiser, 2000). 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