Chapter 23
Cross-LinkingReagents as Toolsfor Identifying Components of the Yeast Mitochondrial Protein Import Machinery PHILIPP E. SCHERER AND UTE C. KRIEG Department of Biochemistry Biocenter, University of Basel CH-4056Easet Switzeriand
I. Introduction 11. Cross-Linking Using Bifunctional Reagents A. Chemical and Physical Properties of Cross-Linking Reagents B. General Considerations C. Identification of a Complex Containing a Mitochondrial 70-kDa Stress Protein and a Partly Translocated Precursor Protein 111. Directed Cross-Linking Using a Photoreactive Mitochondrial Precursor Derivative A. General Considerations B. Identification of a 42-kDa Outer Membrane Protein as a Component of the Mitochondrial Protein Import Site References
I. Introduction Cross-linking approaches have proved useful in identifying pairs of molecules that interact specifically (affinity labeling of receptor-ligand pairs) (e.g., Hanstein, 1979, and references therein) and in characterizing the architecture of multicomponent complexes by establishing nearneighbor relationships (e.g., Capaldi et al., 1979, and references therein). 419 METHODS IN CELL BIOLOGY. VOL. 34
Copyright 01991 by Academic Press. Inc. All rights of reproduction in any form reSeNed.
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Recently. cross-linking techniques have played an important role in identifying proteins that are located adjacent to polypeptides translocating across biological membranes. In the endoplasmic reticulum (ER) system, cross-linking has been successful in identifying the signal sequence receptor (Wiedmann et al., 1987), and mp39. a potential component of the translocation channel across the membrane (Krieg et al., 1989); it has also been instrumental in establishing that the 54-kDa subunit of the signal recognition particle interacts directly with the signal sequence of a nascent polypeptide (Kurzchalia et al., 1986; Krieg et al., 1986). Cross-linking has also been used to characterize a putative mitochondrial receptor for synthetic matrix-targeting peptides (p30) (Gillespie, 1987). As discussed in detail below, Vestweber and colleagues used photocrosslinking of a transiocation-arrested precursor to identify the first membrane component located at the mitochondria1 protein import site (see also Hines and Baker, Chapter 19). Cross-linking approaches can be very powerful in identifying molecules that are located next to each other. When the cross-link forms, the two molecules may exist as a stable complex or may be trapped in a transient state. In the latter case, the experiment requires careful synchronization of the experimental system, so that the cross-linking reaction (usually a low-yield procedure) is carried out in a homogeneous sample. In any case, the formation of a covalent bond between two macromolecules establishes only their spatial proximity, and not necessarily a functional relationship. This latter point must be verified by functional tests, such as competition assays, the use of specific inhibitors. or genetic analysis of the interaction. In principle, individual components of multimeric complexes can also be identified following coimmunoprecipitation with antibodies raised against one of the known constituents. This approach, however, requires that the complex under study remains intact during the time required for immunoprecipitation; in the case of membrane-associated complexes, the structure must also survive solubilization with detergents. Furthermore, the important epitopes recognized by the antibodies must be accessible in the nondissociated complex. Most importantly, coimmunoprecipitation does not allow one to draw conclusions about the molecular arrangement of polypeptides within a larger structure. Cross-linking, on the other hand, can overcome some of these limitations. As convalent bonds are formed, the analysis of a cross-linked product can be carried out under denaturing conditions, directly demonstrating the physical proximity of the two components involved. Depending on the chemistry of the cross-linking reagent. cross-linking may also be sensitive to the functional and/or physical state of a complex, and may thus detect possible conformational changes.
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42 1
11. Cross-Linking Using Bifunctional Reagents A. Chemical and Physical Properties of Cross-Linking Reagents A wide variety of cross-linkers is commercially available (e.g., from Pierce Chemical Company, Rockford, IL 61105). These reagents can be subdivided into two general classes: homobifunctional cross-linkers, which carry two identical functional groups, and heterobifunctional cross-linkers, which carry two reactive groups with different specificities. These later reagents have the advantage that cross-linking can be performed in a stepwise manner. The reactive groups discussed below are most commonly used in protein cross-linking experiments. N-Hydroxysuccinimide esters (NHS esters) react specifically with deprotonated primary amines such as the €-amino group of lysine or the aminoterminal a-amino group of a protein. The reaction is best carried out at slightly alkaline pH and results in the formation of an amide bond between the cross-linking reagent and the protein. The solubility of cross-linkers in water is usually limited, but has been greatly increased by introducing a sulfonate group into the NHS moiety. This offers the possibility of using a membrane-permeant (nonpolar) and a membrane-impermeant (polar) version of a given cross-linker to probe a target for its cis or trans location with respect to a membrane barrier (see below). As NHS esters are quickly hydrolyzed, their solutions should be prepared only immediately before use. Maleimides are widely used for the derivatization of sulfhydryl groups under slightly acidic to neutral conditions. The property to react only with a single type of functional group can be an advantage. On the other hand, this lowers the chance of obtaining a cross-link, as potential reactive groups may not be available at the proper distance and in the proper steric conformation. Photochemical cross-linking reagents are particularly useful, as the cross-linking is performed in two independent steps. (This is in contrast to chemical reagents, which react during the same incubation with both cross-linking moieties.) In a first step, the cross-linker is reacted with a protein via a maleimide or NHS group, as described above. In a second step, the photolabile group is activated by light. Photolysis usually yields strongly electrophilic species that react fairly nonselectively with even the most unreactive groups of organic molecules, such as carbon-hydrogen bonds. Photoactivatable groups include the following reagents: Azides are used, most commonly the aryl azides [for a comprehensive discussion of azide photochemistry, see Bayley and Staros (1984)], which
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PHILIPP E. SCHERER AND UTE C. KRIEG
can be activated at wavelengths above 300 nm. Thus, irradiation is considered nondamaging to protein samples. Illumination initially generates nitrenes, which may subsequently form secondary reactive species. These intermediates react with many functional groups found in polypeptides (this has been shown for C-H, aryl-. N-H, 0-H, and S-H bonds, but others are also predicted). Due to their high reactivity, nitrenes are easily quenched by surrounding water molecules. Azides are very light sensitive and often require handling in safety light. The photoactivation step can also be performed in liquid nitrogen, which is an advantage if a biochemical reaction under investigation needs to be stopped at specific times. Diazirines are another useful class of photoreactive reagents whose photochemistry is relatively well understood (e.g., Brunner, 1989, and references therein). Photoactivation at wavelengths around 350 nm produces highly reactive carbenes. The photoproducts generated by these carbenes are generally more stable during subsequent analysis of the cross-linking experiment than those formed by activated azides. Also, diazirines do not require handling under safety light, but can be used in day light.
B, General Considerations The ability to cross-link two polypeptides depends on two conditions. First, there must be two suitably located target sites on two proteins that can be attacked by an activated moiety of the cross-linker. The more reactive these groups, the higher the chances for the formation of a crosslink. Second, these two target sites must have the right distance from each other. Some flexibility is offered by cross-linkers in which the two reactive groups are separated from each other by spacer arms of different lengths. The longer the spacer. the more distant the partner proteins to be cross-linked can be. One potential problem in probing near-neighbor relationships by crosslinking is abnormal migration of the cross-linked products on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); these products usually migrate more slowly than predicted by the sum of the molecular masses of the individual components. This probably reflects branched structures of these species. The relative ease of obtaining cross-linked products and the difficulty of analyzing such covalent complexes have prompted the development of cross-linkers with a cleavable spacer between the two activated moieties. Such cleavable spacers may contain disulfides that can be reduced using agents such as dithiothreitol or P-mercaptoethanol, diazo groups that can
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be cleaved with dithionite, or several other cleavable functions. Following cross-linking, the sample can be subjected to immunoprecipitation if antibodies against one of the components are available, and the cross-linked product present in the immunoprecipitate can then be cleaved into its components. Finally, these components can be identified by SDS-PAGE. Alternatively, the cross-linked product can be excised from an SDS-PA gel, cleaved, and analyzed on a second SDS-PA gel. Sensitivity of detection can be increased by radioiodinating the proteins contained in the gel slice (Elder et al., 1977). This method allows detection of proteins in the nanogram range. An additional advantage is offered by cleavable cross-linkers whose cleavage transfers a radioactive label to the cross-linked target protein. Such reagents include, e.g., sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3 ‘-dithiopropionate (SASD) and N-[4-(p-azidosalicylamido)butyl]3’(2’-pyridyldithio)propionamide (APDP) ; both are available from Pierce Chemical Co. In general, there is no rule as to which cross-linker will be the one of choice for a particular problem. Success is a matter of trial and error. Still, it usually pays to consider the chemical properties of the cross-linker and the probable location of the putative partner protein(s). The following examples demonstrate the use of cross-linking reagents in detecting proteins that catalyze the import of precursor proteins into mitochondria.
C. Identification of a Complex Containing a Mitochondria1 70-kDa Stress Protein and a Partly Translocated Precursor Protein To identify protein components that may play a role in the mitochondria1 import of precursor proteins, mitochondria were first allowed to accumulate partially translocated precursor molecules. Translocation was arrested by attaching a tightly folded bovine pancreatic trypsin inhibitor (BPTI) moiety to the carboxy-terminal cysteine residue of a purified authentic precursor protein (Vestweber and Schatz, 1988). When the mitochondria were then treated with the homobifunctional, membrane-permeable crosslinker dithiobis (succinimidopropionate) (DSP), two high-molecular-mass products were formed that contained the stuck precursor and at least one additional protein (Scherer et al., 1990). The cross-linked complexes were also obtained after solubilization of the membranes in Triton X-100. However, if the membrane-impermeant analog of DSP, 3,3’-dithiobis(sulfosuccinimidy1)propionate (DTSSP), was used, the cross-links were only obtained after solubilization and not with intact mitochondria. This
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suggested that the target molecule was located in the matrix space. The approximate molecular mass of the cross-linked protein was assessed by iodinating mitochondria, after accumulation of the stuck precursor and cross-linking, with the membrane-permeant Bolton-Hunter reagent (Bolton and Hunter, 1973). The cross-linked complexes were immunoprecipitated with antiserum raised against the precursor protein, and then analyzed by SDS-PAGE. The bands of interest were excised from the gel, the cross-linker was cleaved by the addition of DTT, and the products were analyzed on a second SDS-PA gel. This revealed that the precursor protein had been cross-linked to a 70-kDa protein. In order to isolate this protein and to study its function, we raised antibodies against 70-kDa mitochondrial proteins and tested them for their ability to immunoprecipitate the cross-linked product. After affinity purification of the sera against mitochondrial proteins that had been separated by anion-exchange chromatography, we obtained a monospecific antibody preparation. These antibodies were used to confirm the submitochondrial location of the cross-linked 70-kDa protein using immunoelectron microscopy. This led to the unambigous identification of the 70-kDa protein as the mitochondrial 70-kDa stress protein, the gene product of the nuclear SSCl gene (Craig et al., 1987).
111. Directed Cross-Linking Using a Photoreactive Mitochondria1 Precursor Derivative
A. General Considerations As an alternative to using cross-linking reagents that are added to the sample after the desired functional state of a given system has been achieved, one can use (photo)chemical derivatives of a molecule that is itself part of the structure under investigation. This greatly increases the efficiency and specificity of cross-linking. However, it can also cause major problems. as the derivatized macromolecule may no longer be functional. This point must be checked in all such cross-linking studies.
B. Identification of a 42-kDa Outer Membrane Protein as a Component of the Mitochondrial Protein Import Site Vestweber et al. (1989) used a chimeric mitochondrial fusion protein derivatized with a trifunctional cross-linker to identify an outer membrane protein that was located in close proximity to a translocation-arrested precursor. This outer membrane protein (termed ISP42) was subsequently
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shown to be essential for the import of precursor molecules and for cell viability (Baker et al., 1990). Each of the cross-linker’s three functional groups could be activated individually. Two of these groups were sequentially reacted to Iink the precursor protein to BPTI. (BPTI caused the product to become stuck across the two mitochondrial membranes; see above.) First, the NHS group was used to derivatize BPTI. After removal of excess cross-linker by gel filtration, the derivatized BPTI was attached via one of its maleimide groups to the unique carboxy-terminal cysteine of the DV12 variant of the cytochrome oxidase subunit IV-dihydrofolate reductase (COXIV-DHFR) hybrid precursor (see Krieg and Scherer, Chapter 22). ’When the resulting COXIV-DHFR-BPTI adduct was then incubated with mitochondria, it jammed the mitochondrial protein import sites. At this stage, the third, photoreactive diazirine group of the crosslinker was activated. The photocross-linking reaction proceeded with high efficiency (almost 10% of all stuck precursor became cross-linked to the same target molecule) and resulted in a convalent complex of the precursor with a 42-kDa protein. The success of these two studies can most likely be attributed to the design of the experimental system. The goal was to work with a synchronized population of imported precursor molecules. The “stuck precursor” approach provided the basis for examining the molecular environment of a precursor in transit across the two mitochondrial membranes from two different angles. First, the directed cross-linking (with the photoreactive group being a part of the investigated system) yielded a single target molecule; this reflects the close physical proximity of the carboxy terminus of precursor and ISP42. No other proteins were cross-linked to the stuck precursor, either because ISP42 was the only neighbor molecule, or, more likely, because of steric constraints of the cross-linker; other proteins may simply have been too far away. Second, using a small soluble chemical cross-linker in the same system resulted in the identification of one major covalent complex that involved a protein of the mitochondrial matrix. Again, no other proteins were cross-linked to the stuck precursor. This is probably explained by the fact that binding of the stuck precursor to the 70-kDa stress protein locks the position of the imported protein and allows little flexibility for cross-linkingto more distant proteins. In addition, crosslink formation requires two reactive €-amino groups in a defined steric conformation. The two cross-linking techniques give completely different, although complementary, information about the environment around different domains of the same precursor. These examples document the possibilities, and the limitations, of applying cross-linking techniques to membrane systems.
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REFERENCES Baker. K. P., Schaniel, A., Vestweber, D.. and Schatz, G. (1990). A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature (London) 348,605-609. Bayley, H.. and Staros. J . V. (1984). Photoaffinity labeling and related techniques. In “Azides and Nitrenes: Reactivity and Utility” (E. F. V. Scriven. ed.), pp, 433-490. Academic Press, Orlando, Florida. Bolton. A . E., and Hunter, W. M. (1973). The labelling of proteins to high specific radioactivities by conjugation to a ’251-containingacylating agent. Biochem. J . 133, 529-539. Brunner, J . (1989). Photochemical labeling of apolar phase of membranes. In “Methods in Enzymology” (S. Fleischer and B. Fleischer, eds.), Vol. 172, pp. 628-687. Academic Press, San Diego, California. Capaldi. R . A., Briggs, M. M., and Smith. R. J . (1979). Cleavable bifunctional reagents for studying near neighbor relationships among mitochondrial inner membrane complexes. In “Methods in Enzymology” (S. Fleischer and L. Packer, eds.), Vol. 56, pp. 630-642. Academic Press, New York. Craig, E. A.. Kramer, J . , and Kosic-Smithers, J . (1987). SSCl, a member of the 70-kDa heat shock protein multigene family of Succhuroyrnces cerevisiue, is essential for growth. Proc. Nutl. Acad. Sci. U.S. A . 84, 4156-4160. Elder. J . H., Pickett, R. A., 11, Hampton, .I.and . Lerner, R. A. (1977). Radioiodination of proteins in single polyacrylamide gel slices. J . Biol. Chem. 252, 6510-6515. Gillespie, L. (1987). Identification of an outer mitochondrial membrane protein that interacts with a synthetic signal peptide. J . Biol. Chern. 262, 7939-7942. Hanstein, W. G. (1979). Photoaffinity labeling of membrane components. In “Methods in Enzymology” (S. Fleischer and L. Packer. eds.), Vol. 56, pp. 653-683. Academic Press, New York. Krieg, U . C . . Walter, P., and Johnson, A. E. (1986). Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle. Proc. Nutl. Acud. Sci. U . S. A . 83, 8604-8608. Krieg, U. C.. Johnson, A. E., and Walter. P. (1989). Protein translocation across the endoplasmic reticulum membrane: Identification by photocrosslinking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel. 1.Cell Bio. 109, 20332043. Kurzchalia. T. V . , Wiedmann, M., Gishovich, A. S., Bochkarva, E. S., Bielka, H., and Rapoport, T. A. (1986). The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. Nufure (London) 320, 634-636. Scherer, P. E., Krieg, U . C . , Hwang, S. T., Vestweber, D.. and Schatz, G. (1990). A precursor protein partly translocated into yeast mitochondria is bound to a 70 kDa mitochondrial stress protein. EMBO J . 9, 4315-4322. Vestweber, D., and Schatz, G. (1988). A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites. J Cell Biol. 107, 2037-2043. Vestweber, D., Brunner, J., Baker, A , , and Schatz, G . (1989). A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site. Nature (London) 341, 205-209. Wiedmann, M., Kurzchalia, T. V., Hartmann. E., and Rapoport, T. A , , (1987). A signal sequence receptor in the endoplasmic reticulum membrane. Nufure (London) 328, 830833.
Cross-LinkingReagents as Toolsfor Identifying Components of the Yeast Mitochondrial Protein Import Machinery PHILIPP E. SCHERER AND UTE C. KRIEG Department of Biochemistry Biocenter, University of Basel CH-4056Easet Switzeriand
I. Introduction 11. Cross-Linking Using Bifunctional Reagents A. Chemical and Physical Properties of Cross-Linking Reagents B. General Considerations C. Identification of a Complex Containing a Mitochondrial 70-kDa Stress Protein and a Partly Translocated Precursor Protein 111. Directed Cross-Linking Using a Photoreactive Mitochondrial Precursor Derivative A. General Considerations B. Identification of a 42-kDa Outer Membrane Protein as a Component of the Mitochondrial Protein Import Site References
I. Introduction Cross-linking approaches have proved useful in identifying pairs of molecules that interact specifically (affinity labeling of receptor-ligand pairs) (e.g., Hanstein, 1979, and references therein) and in characterizing the architecture of multicomponent complexes by establishing nearneighbor relationships (e.g., Capaldi et al., 1979, and references therein). 419 METHODS IN CELL BIOLOGY. VOL. 34
Copyright 01991 by Academic Press. Inc. All rights of reproduction in any form reSeNed.
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Recently. cross-linking techniques have played an important role in identifying proteins that are located adjacent to polypeptides translocating across biological membranes. In the endoplasmic reticulum (ER) system, cross-linking has been successful in identifying the signal sequence receptor (Wiedmann et al., 1987), and mp39. a potential component of the translocation channel across the membrane (Krieg et al., 1989); it has also been instrumental in establishing that the 54-kDa subunit of the signal recognition particle interacts directly with the signal sequence of a nascent polypeptide (Kurzchalia et al., 1986; Krieg et al., 1986). Cross-linking has also been used to characterize a putative mitochondrial receptor for synthetic matrix-targeting peptides (p30) (Gillespie, 1987). As discussed in detail below, Vestweber and colleagues used photocrosslinking of a transiocation-arrested precursor to identify the first membrane component located at the mitochondria1 protein import site (see also Hines and Baker, Chapter 19). Cross-linking approaches can be very powerful in identifying molecules that are located next to each other. When the cross-link forms, the two molecules may exist as a stable complex or may be trapped in a transient state. In the latter case, the experiment requires careful synchronization of the experimental system, so that the cross-linking reaction (usually a low-yield procedure) is carried out in a homogeneous sample. In any case, the formation of a covalent bond between two macromolecules establishes only their spatial proximity, and not necessarily a functional relationship. This latter point must be verified by functional tests, such as competition assays, the use of specific inhibitors. or genetic analysis of the interaction. In principle, individual components of multimeric complexes can also be identified following coimmunoprecipitation with antibodies raised against one of the known constituents. This approach, however, requires that the complex under study remains intact during the time required for immunoprecipitation; in the case of membrane-associated complexes, the structure must also survive solubilization with detergents. Furthermore, the important epitopes recognized by the antibodies must be accessible in the nondissociated complex. Most importantly, coimmunoprecipitation does not allow one to draw conclusions about the molecular arrangement of polypeptides within a larger structure. Cross-linking, on the other hand, can overcome some of these limitations. As convalent bonds are formed, the analysis of a cross-linked product can be carried out under denaturing conditions, directly demonstrating the physical proximity of the two components involved. Depending on the chemistry of the cross-linking reagent. cross-linking may also be sensitive to the functional and/or physical state of a complex, and may thus detect possible conformational changes.
23.
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42 1
11. Cross-Linking Using Bifunctional Reagents A. Chemical and Physical Properties of Cross-Linking Reagents A wide variety of cross-linkers is commercially available (e.g., from Pierce Chemical Company, Rockford, IL 61105). These reagents can be subdivided into two general classes: homobifunctional cross-linkers, which carry two identical functional groups, and heterobifunctional cross-linkers, which carry two reactive groups with different specificities. These later reagents have the advantage that cross-linking can be performed in a stepwise manner. The reactive groups discussed below are most commonly used in protein cross-linking experiments. N-Hydroxysuccinimide esters (NHS esters) react specifically with deprotonated primary amines such as the €-amino group of lysine or the aminoterminal a-amino group of a protein. The reaction is best carried out at slightly alkaline pH and results in the formation of an amide bond between the cross-linking reagent and the protein. The solubility of cross-linkers in water is usually limited, but has been greatly increased by introducing a sulfonate group into the NHS moiety. This offers the possibility of using a membrane-permeant (nonpolar) and a membrane-impermeant (polar) version of a given cross-linker to probe a target for its cis or trans location with respect to a membrane barrier (see below). As NHS esters are quickly hydrolyzed, their solutions should be prepared only immediately before use. Maleimides are widely used for the derivatization of sulfhydryl groups under slightly acidic to neutral conditions. The property to react only with a single type of functional group can be an advantage. On the other hand, this lowers the chance of obtaining a cross-link, as potential reactive groups may not be available at the proper distance and in the proper steric conformation. Photochemical cross-linking reagents are particularly useful, as the cross-linking is performed in two independent steps. (This is in contrast to chemical reagents, which react during the same incubation with both cross-linking moieties.) In a first step, the cross-linker is reacted with a protein via a maleimide or NHS group, as described above. In a second step, the photolabile group is activated by light. Photolysis usually yields strongly electrophilic species that react fairly nonselectively with even the most unreactive groups of organic molecules, such as carbon-hydrogen bonds. Photoactivatable groups include the following reagents: Azides are used, most commonly the aryl azides [for a comprehensive discussion of azide photochemistry, see Bayley and Staros (1984)], which
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PHILIPP E. SCHERER AND UTE C. KRIEG
can be activated at wavelengths above 300 nm. Thus, irradiation is considered nondamaging to protein samples. Illumination initially generates nitrenes, which may subsequently form secondary reactive species. These intermediates react with many functional groups found in polypeptides (this has been shown for C-H, aryl-. N-H, 0-H, and S-H bonds, but others are also predicted). Due to their high reactivity, nitrenes are easily quenched by surrounding water molecules. Azides are very light sensitive and often require handling in safety light. The photoactivation step can also be performed in liquid nitrogen, which is an advantage if a biochemical reaction under investigation needs to be stopped at specific times. Diazirines are another useful class of photoreactive reagents whose photochemistry is relatively well understood (e.g., Brunner, 1989, and references therein). Photoactivation at wavelengths around 350 nm produces highly reactive carbenes. The photoproducts generated by these carbenes are generally more stable during subsequent analysis of the cross-linking experiment than those formed by activated azides. Also, diazirines do not require handling under safety light, but can be used in day light.
B, General Considerations The ability to cross-link two polypeptides depends on two conditions. First, there must be two suitably located target sites on two proteins that can be attacked by an activated moiety of the cross-linker. The more reactive these groups, the higher the chances for the formation of a crosslink. Second, these two target sites must have the right distance from each other. Some flexibility is offered by cross-linkers in which the two reactive groups are separated from each other by spacer arms of different lengths. The longer the spacer. the more distant the partner proteins to be cross-linked can be. One potential problem in probing near-neighbor relationships by crosslinking is abnormal migration of the cross-linked products on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); these products usually migrate more slowly than predicted by the sum of the molecular masses of the individual components. This probably reflects branched structures of these species. The relative ease of obtaining cross-linked products and the difficulty of analyzing such covalent complexes have prompted the development of cross-linkers with a cleavable spacer between the two activated moieties. Such cleavable spacers may contain disulfides that can be reduced using agents such as dithiothreitol or P-mercaptoethanol, diazo groups that can
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423
be cleaved with dithionite, or several other cleavable functions. Following cross-linking, the sample can be subjected to immunoprecipitation if antibodies against one of the components are available, and the cross-linked product present in the immunoprecipitate can then be cleaved into its components. Finally, these components can be identified by SDS-PAGE. Alternatively, the cross-linked product can be excised from an SDS-PA gel, cleaved, and analyzed on a second SDS-PA gel. Sensitivity of detection can be increased by radioiodinating the proteins contained in the gel slice (Elder et al., 1977). This method allows detection of proteins in the nanogram range. An additional advantage is offered by cleavable cross-linkers whose cleavage transfers a radioactive label to the cross-linked target protein. Such reagents include, e.g., sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3 ‘-dithiopropionate (SASD) and N-[4-(p-azidosalicylamido)butyl]3’(2’-pyridyldithio)propionamide (APDP) ; both are available from Pierce Chemical Co. In general, there is no rule as to which cross-linker will be the one of choice for a particular problem. Success is a matter of trial and error. Still, it usually pays to consider the chemical properties of the cross-linker and the probable location of the putative partner protein(s). The following examples demonstrate the use of cross-linking reagents in detecting proteins that catalyze the import of precursor proteins into mitochondria.
C. Identification of a Complex Containing a Mitochondria1 70-kDa Stress Protein and a Partly Translocated Precursor Protein To identify protein components that may play a role in the mitochondria1 import of precursor proteins, mitochondria were first allowed to accumulate partially translocated precursor molecules. Translocation was arrested by attaching a tightly folded bovine pancreatic trypsin inhibitor (BPTI) moiety to the carboxy-terminal cysteine residue of a purified authentic precursor protein (Vestweber and Schatz, 1988). When the mitochondria were then treated with the homobifunctional, membrane-permeable crosslinker dithiobis (succinimidopropionate) (DSP), two high-molecular-mass products were formed that contained the stuck precursor and at least one additional protein (Scherer et al., 1990). The cross-linked complexes were also obtained after solubilization of the membranes in Triton X-100. However, if the membrane-impermeant analog of DSP, 3,3’-dithiobis(sulfosuccinimidy1)propionate (DTSSP), was used, the cross-links were only obtained after solubilization and not with intact mitochondria. This
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PHILIPP E. SCHERER AND UTE C. KRIEG
suggested that the target molecule was located in the matrix space. The approximate molecular mass of the cross-linked protein was assessed by iodinating mitochondria, after accumulation of the stuck precursor and cross-linking, with the membrane-permeant Bolton-Hunter reagent (Bolton and Hunter, 1973). The cross-linked complexes were immunoprecipitated with antiserum raised against the precursor protein, and then analyzed by SDS-PAGE. The bands of interest were excised from the gel, the cross-linker was cleaved by the addition of DTT, and the products were analyzed on a second SDS-PA gel. This revealed that the precursor protein had been cross-linked to a 70-kDa protein. In order to isolate this protein and to study its function, we raised antibodies against 70-kDa mitochondrial proteins and tested them for their ability to immunoprecipitate the cross-linked product. After affinity purification of the sera against mitochondrial proteins that had been separated by anion-exchange chromatography, we obtained a monospecific antibody preparation. These antibodies were used to confirm the submitochondrial location of the cross-linked 70-kDa protein using immunoelectron microscopy. This led to the unambigous identification of the 70-kDa protein as the mitochondrial 70-kDa stress protein, the gene product of the nuclear SSCl gene (Craig et al., 1987).
111. Directed Cross-Linking Using a Photoreactive Mitochondria1 Precursor Derivative
A. General Considerations As an alternative to using cross-linking reagents that are added to the sample after the desired functional state of a given system has been achieved, one can use (photo)chemical derivatives of a molecule that is itself part of the structure under investigation. This greatly increases the efficiency and specificity of cross-linking. However, it can also cause major problems. as the derivatized macromolecule may no longer be functional. This point must be checked in all such cross-linking studies.
B. Identification of a 42-kDa Outer Membrane Protein as a Component of the Mitochondrial Protein Import Site Vestweber et al. (1989) used a chimeric mitochondrial fusion protein derivatized with a trifunctional cross-linker to identify an outer membrane protein that was located in close proximity to a translocation-arrested precursor. This outer membrane protein (termed ISP42) was subsequently
23.
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shown to be essential for the import of precursor molecules and for cell viability (Baker et al., 1990). Each of the cross-linker’s three functional groups could be activated individually. Two of these groups were sequentially reacted to Iink the precursor protein to BPTI. (BPTI caused the product to become stuck across the two mitochondrial membranes; see above.) First, the NHS group was used to derivatize BPTI. After removal of excess cross-linker by gel filtration, the derivatized BPTI was attached via one of its maleimide groups to the unique carboxy-terminal cysteine of the DV12 variant of the cytochrome oxidase subunit IV-dihydrofolate reductase (COXIV-DHFR) hybrid precursor (see Krieg and Scherer, Chapter 22). ’When the resulting COXIV-DHFR-BPTI adduct was then incubated with mitochondria, it jammed the mitochondrial protein import sites. At this stage, the third, photoreactive diazirine group of the crosslinker was activated. The photocross-linking reaction proceeded with high efficiency (almost 10% of all stuck precursor became cross-linked to the same target molecule) and resulted in a convalent complex of the precursor with a 42-kDa protein. The success of these two studies can most likely be attributed to the design of the experimental system. The goal was to work with a synchronized population of imported precursor molecules. The “stuck precursor” approach provided the basis for examining the molecular environment of a precursor in transit across the two mitochondrial membranes from two different angles. First, the directed cross-linking (with the photoreactive group being a part of the investigated system) yielded a single target molecule; this reflects the close physical proximity of the carboxy terminus of precursor and ISP42. No other proteins were cross-linked to the stuck precursor, either because ISP42 was the only neighbor molecule, or, more likely, because of steric constraints of the cross-linker; other proteins may simply have been too far away. Second, using a small soluble chemical cross-linker in the same system resulted in the identification of one major covalent complex that involved a protein of the mitochondrial matrix. Again, no other proteins were cross-linked to the stuck precursor. This is probably explained by the fact that binding of the stuck precursor to the 70-kDa stress protein locks the position of the imported protein and allows little flexibility for cross-linkingto more distant proteins. In addition, crosslink formation requires two reactive €-amino groups in a defined steric conformation. The two cross-linking techniques give completely different, although complementary, information about the environment around different domains of the same precursor. These examples document the possibilities, and the limitations, of applying cross-linking techniques to membrane systems.
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