The Hydrophilic Domain of Tic110, an Inner Envelope Membrane Component of the Chloroplastic Protein Translocation Apparatus, Faces the Stromal Compartment*

It has previously been found that Tic110, an integral protein of the chloroplast inner envelope membrane, is a component of the chloroplastic protein import apparatus. However, conflicting reports exist concerning the topology of this protein within the inner envelope membrane. In this report, we provide evidence that indicates that the large (>90-kDa) hydrophilic domain of Tic110 is localized within the chloroplast stroma. Trypsin, a protease that cannot penetrate the permeability barrier of the inner envelope membrane, degrades neither Tic110 nor other proteins exposed to the stromal compartment but is able to digest proteins exposed to the intermembrane space between the two envelope membranes. Previous reports indicating that trypsin is able to degrade Tic110 were influenced by incomplete quenching of protease activity. When trypsin is not sufficiently quenched, it is able to digest Tic110, but only after chloroplasts have been ruptured. It is therefore necessary to employ adequate quenching protocols, such as the one reported here, whenever trypsin is utilized as an analytical tool. Based on a stromal localization for the majority of Tic110, we propose that this protein may be involved in the recruitment of stromal factors, possibly molecular chaperones, to the translocation apparatus during protein import.

It has previously been found that Tic110, an integral protein of the chloroplast inner envelope membrane, is a component of the chloroplastic protein import apparatus. However, conflicting reports exist concerning the topology of this protein within the inner envelope membrane. In this report, we provide evidence that indicates that the large (>90-kDa) hydrophilic domain of Tic110 is localized within the chloroplast stroma. Trypsin, a protease that cannot penetrate the permeability barrier of the inner envelope membrane, degrades neither Tic110 nor other proteins exposed to the stromal compartment but is able to digest proteins exposed to the intermembrane space between the two envelope membranes. Previous reports indicating that trypsin is able to degrade Tic110 were influenced by incomplete quenching of protease activity. When trypsin is not sufficiently quenched, it is able to digest Tic110, but only after chloroplasts have been ruptured. It is therefore necessary to employ adequate quenching protocols, such as the one reported here, whenever trypsin is utilized as an analytical tool. Based on a stromal localization for the majority of Tic110, we propose that this protein may be involved in the recruitment of stromal factors, possibly molecular chaperones, to the translocation apparatus during protein import.
The majority of chloroplastic proteins are encoded within the nuclei of plant cells and are synthesized on cytoplasmic ribosomes. As a result, these proteins must be imported into the chloroplast post-translationally, usually via an unfolded, higher molecular weight precursor form containing a N-terminal transit peptide (1)(2)(3)(4). Once a precursor protein has entered the chloroplast stroma, the transit peptide is cleaved off by the stromal processing peptidase, and the protein is folded into its mature form (4). Protein import into chloroplasts is mediated by a proteinaceous translocation apparatus that spans both the outer and inner envelope membranes of this organelle. Several components of the translocation apparatus have been identified, including the outer membrane components Toc86, 1 Toc75, and Toc34; an inner membrane component, Tic110; and a primarily stromal component, ClpC, an hsp100 homologue (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).
The first component of the inner envelope membrane translocation apparatus to be cloned was Tic110. Using chemical cross-linking and coimmunoprecipitation techniques, two separate laboratories have found Tic110 in a complex with both a translocating precursor and components of the outer membrane translocation apparatus (13,14). Tic110 is an integral protein of the inner envelope membrane of chloroplasts, with either one or two putative, hydrophobic, transmembrane domains located near its N terminus (13,14). The overall topology of Tic110 within the inner envelope membrane, however, remains a point of debate. Lü beck et al. (14) reported that Tic110 spans the membrane once and that its large (Ͼ90-kDa) hydrophilic domain is oriented toward the intermembrane space between the outer and inner envelope membranes. On the other hand, Kessler and Blobel (13) proposed that Tic110 spans the membrane twice and that its hydrophilic domain is contained within the chloroplast stroma. To date, no evidence has been presented that satisfactorily resolves this controversy.
Knowing the topology of Tic110 will be important in assigning a putative function to this protein. For instance, if Tic110 is oriented toward the chloroplast intermembrane space, then it may function by interacting with the outer membrane translocation apparatus, promoting the formation of contact sites between the two envelope membranes (14). However, if Tic110 is instead exposed to the stromal compartment, then it is more likely that the protein acts by recruiting stromal proteins, for example molecular chaperones, to the protein import apparatus (13).
In previous studies, the topology of Tic110 was investigated by analyzing the protease sensitivity of the protein within intact chloroplasts, a technique that has been used for other chloroplastic membrane proteins and for membrane proteins of other organelles (7,11,13,14,17,18). Two of the most widely used proteases in such studies are thermolysin and trypsin. Thermolysin has been used to selectively degrade outer envelope membrane proteins exposed on the surface of chloroplasts, since this protease, at moderate concentrations, does not penetrate the outer membrane (19). Trypsin, however, does penetrate the chloroplast outer envelope membrane, but it does not, at moderate concentrations, destroy the permeability barrier of the inner membrane (19 -21). Thus, trypsin is useful in defining the topology of inner envelope membrane proteins and in localizing soluble proteins to the intermembrane space of the chloroplast.
In this paper, we report on the topology of Tic110, attempting to resolve the controversy that currently exists concerning the orientation of this protein within the chloroplast inner envelope membrane. When steps are taken to adequately quench proteases, Tic110 is degraded by neither trypsin nor thermolysin, indicating that the large hydrophilic domain of Tic110 is contained within the chloroplast stromal compartment. In addition, when trypsin is insufficiently quenched, Tic110 is degraded, but only after chloroplasts are broken open. Comparison of the protease sensitivity of Tic110 with those of proteins of established topology lends further support to the conclusion that Tic110 is indeed oriented toward the chloroplast stroma.
Trypsin Digestion of Intact Chloroplasts-Purified intact chloroplasts (50 g of chlorophyll) were incubated with trypsin (6300 BAEE units/mg; 10 -1000 g of trypsin/mg of chlorophyll) in import buffer containing calcium chloride at a final concentration of 0.1 mM. The final reaction volume for these digestions was 300 l. After incubation with the protease for either 10 min or 60 min at room temperature, trypsin activity was quenched by adding either PMSF at a final concentration of 1 mM or by adding a mixture of protease inhibitors to a final concentration of 1 mM PMSF, 0.05 mg/ml TLCK, 0.1 mg/ml soybean trypsin inhibitor, and 2 g/ml aprotinin. Chloroplasts were incubated with the quenching reagents for 10 min on ice.
After quenching, intact chloroplasts were reisolated over a 40% (v/v) Percoll cushion. The recovered chloroplasts were lysed hypotonically and fractionated into crude membrane and soluble fractions as described previously (22), except that the lysis buffer contained either PMSF at a final concentration of 1 mM or a protease inhibitor mixture at final concentrations of 5 mM PMSF, 0.05 mg/ml TLCK, and 0.1 mg/ml soybean trypsin inhibitor. Fractions were analyzed by SDS-PAGE and immunoblotting with either Tic110 or Toc75 antibodies essentially as described by Tranel et al. (12). Variations to this protocol are given in the figure legends.
One sample from each import reaction was lysed hypotonically, as described previously (22), without any further treatment. A second sample was incubated on ice for 30 min with 0.2 mg/ml thermolysin. Protease activity was quenched by adding EDTA to a final concentration of 5 mM. Intact chloroplasts were then reisolated over a 40% (v/v) Percoll cushion containing 5 mM EDTA and lysed hypotonically. The remaining two samples from each import reaction were incubated in the presence of trypsin (6000 BAEE units/mg) at a concentration of 500 g of trypsin/mg of chlorophyll for 60 min at room temperature. Trypsin activity was quenched for 10 min on ice either with PMSF at a final concentration of 1 mM or with a mixture of protease inhibitors at final concentrations of 1 mM PMSF, 0.05 mg/ml TLCK, 0.1 mg/ml soybean trypsin inhibitor, and 2 g/ml aprotinin. Intact chloroplasts from the two trypsin treatments were then reisolated and lysed as described above. Lysed chloroplasts from all four treatments were fractionated into crude membrane and soluble fractions as described by Bruce et al. (22). The protein concentration of each fraction was determined by the Lowry protein assay (25). Equal amounts of protein from each fraction were analyzed by SDS-PAGE and fluorography or immunoblotting (12).
Trypsin Digestion of Purified Inner Envelope Membrane Vesicles-Inner envelope membranes were purified from intact chloroplasts es-sentially as described by Keegstra and Yousif (26), except that the purified inner membranes were resuspended in lysis buffer (25 mM HEPES-KOH (pH 8.0), 4 mM MgCl 2 ) at a concentration of 0.5 mg/ml. Protein concentration was determined by the Bradford protein assay (Bio-Rad). Purified inner envelope membranes (20 g of protein) were incubated with trypsin (6000 BAEE units/mg; 1-10,000 ng of trypsin/mg of protein) in the presence of 0.1 mM calcium chloride for 10 min at room temperature. The final reaction volume for these digestions was 200 l. Trypsin activity was quenched by the addition of a mixture of protease inhibitors at final concentrations of 1 mM PMSF, 0.05 mg/ml TLCK, 0.1 mg/ml trypsin inhibitor, and 2 g/ml aprotinin. The quenched reactions were incubated for 10 min on ice. Inner envelope membranes were then recovered by centrifuging the samples at 250,000 ϫ g for 10 min. Samples were analyzed by SDS-PAGE and immunoblotting with antibodies against either Tic110 or ClpC as described previously (12).
Antibodies-All antibodies used in this investigation were polyclonal and raised in rabbits. Antiserum to Tic110 was generated as described by Akita et al. (15). Antiserum against Toc75 was raised as discussed by Tranel et al. (12). Antiserum to Toc34 (9) was a gift from D. Schnell. Affinity-purified anti-ClpC antibodies (27) were a gift from J. Shanklin.

Tic110 Is Resistant to Digestion by Adequately Quenched
Trypsin-It has been reported that certain proteases, most notably trypsin, are able to destroy the permeability barrier of the outer envelope membrane of chloroplasts and thereby degrade outer membrane proteins, as well as inner envelope membrane proteins exposed to the intermembrane space, while leaving stromally exposed proteins undigested (19 -21). Consequently, this method can be used to selectively degrade inner envelope membrane proteins that are oriented toward the intermembrane space while leaving stromally exposed inner membrane proteins intact. Such selective proteolysis techniques have previously been utilized to analyze the location and topology of various chloroplast envelope membrane proteins, including Tic110 (13,14,18).
During efforts to repeat and extend these previous studies, we observed that Tic110 was resistant to degradation when intact chloroplasts were incubated with a range of trypsin concentrations (data not shown), indicating that this protein was not exposed to the chloroplast intermembrane space. These results were in direct contrast with the trypsin sensitivity of Tic110 reported by Lü beck et al. (14). However, several differences in protocol existed between the two experiments, including the length of time used for trypsin digestion and the reagents used to quench trypsin activity. Consequently, we sought to determine whether these protocol differences could explain the contrasting results.
Intact chloroplasts were incubated with trypsin for either 10 min (Fig. 1, lanes 1-3 and lanes 7-9) or for 60 min (Fig. 1, lanes  4 and 5 and lanes 10 and 11), as described by Lü beck et al. (14).  lanes 6 and 12) was 500 g of protease/mg of chlorophyll. Trypsin activity was quenched as indicated either after (lanes 2-5 and 8 -11) or before (lanes 6 and 12) trypsin addition. Intact chloroplasts were reisolated, lysed, and separated into membrane and soluble protein fractions. Equivalent volumes of each membrane protein fraction were analyzed by SDS-PAGE and immunoblotting with antibodies against either Tic110 or Toc75.
After digestion for the specified period of time, trypsin activity was quenched either with a mixture of protease inhibitors (Fig.  1, lanes 1-5) or with 1 mM PMSF (Fig. 1, lanes 7-11), as reported by Lü beck et al. (14). Degradation of Tic110 was not significantly affected by the duration of incubation with the protease (Fig. 1, compare lanes 2 and 3 with lanes 4 and 5). On the other hand, the quench protocol had a dramatic effect on Tic110 digestion (Fig. 1, compare lanes 2-5 and lanes 8 -11). Tic110 remained undigested when trypsin was quenched with the mixture of protease inhibitors but was completely degraded when 1 mM PMSF was used to quench trypsin activity. These observations indicated that 1 mM PMSF was insufficient to quench protease activity. This result was supported by the finding that when chloroplasts were incubated with 1 mM PMSF prior to trypsin addition, Tic110 was still completely degraded (Fig. 1, lane 12). Other differences (i.e. number of washes) between our protease digestion protocol and that of Lü beck et al. (14) were also tested for their effects on Tic110 degradation. However, none affected the pattern of Tic110 digestion (data not shown). We concluded, therefore, that the difference in results could be completely explained by differences in the methods used to quench trypsin activity.
Tic110 Is Degraded by Insufficiently Quenched Trypsin only after Chloroplast Lysis-We next sought to determine at what stage of the protease digestion protocol trypsin degraded Tic110 when 1 mM PMSF was used as the quenching reagent. Specifically, we wanted to determine whether Tic110 was degraded before chloroplast lysis, when the permeability barrier of the inner membrane was still intact, or after lysis, when the inner membrane had been ruptured. There were three stages during our protocol in which degradation of Tic110 by trypsin could occur: before chloroplasts were broken open (incubation of chloroplasts with trypsin, quenching of protease activity, and reisolation of intact chloroplasts), during chloroplast lysis, or during postlysis steps (membrane sedimentation and incubation of the membranes in SDS-PAGE sample buffer). To distinguish among these possibilities, we quenched trypsin-treated chloroplasts with the mixture of protease inhibitors before lysis, during lysis, and/or after lysis. During those steps when the protease inhibitor mixture was not added, 1 mM PMSF was added in its place. Fig. 2 shows the results from this experimental approach. When the quench mixture was added at all three stages or just during and after lysis, Tic110 was not significantly degraded ( Fig. 2A, lanes 1 and 2). Tic110 was completely digested only when the quenching mixture was added just during the postlysis stage (Fig. 2A, lane 3), indicating that it was most likely degraded by active trypsin during chloroplast lysis. In addition, as long as the protease inhibitor mixture was added before and/or during chloroplast lysis, Tic110 was not digested by trypsin (Fig. 2B). Thus, it appeared that unless trypsin was adequately quenched before or at the time of lysis, Tic110 was digested by the protease once chloroplasts were broken open.
Trypsin Degrades Proteins Exposed to the Intermembrane Space but Not Tic110 -Recently, several investigators have utilized protease digestion techniques to analyze the location and topology of newly imported chloroplastic proteins (18,23). We obtained these precursor constructs in order to determine whether the quenching protocol used had an effect on the results and to compare the protease sensitivity of constructs with known topology to that of Tic110 constructs. Intact chloroplasts were subjected to an import assay with one of five different precursor proteins: prToc75 (12); tp110 -110N, a truncated version of prTic110 containing the putative transmembrane domain(s) and approximately one-fifth (Ͻ20 kDa) of the hydrophilic domain (23); tpSS-110N, a chimeric precursor containing the transit peptide of the small subunit of ribulose-1,5bisphosphate carboxylase (SS) attached to the truncated version of mature Tic110 (23); tpToc75-mSS, a chimeric precursor consisting of the transit peptide of Toc75 attached to the mature form of SS (18); and prSS (24). After import, intact chloroplasts were reisolated and digested with either thermolysin or trypsin. Trypsin-treated chloroplasts were quenched with either the protease inhibitor mixture (trypsin I protocol) or 1 mM PMSF (trypsin II protocol). Following protease digestion, intact chloroplasts were reisolated, lysed, and separated into membrane and soluble protein fractions. The proteins from these fractions were then analyzed by SDS-PAGE and fluorography to detect the newly imported, radiolabeled proteins (Fig.  3A) or immunoblotting to detect endogenous proteins (Fig. 3B).
The processed forms of prToc75 (mToc75 and iToc75), which were used as markers for the outer envelope membrane, were degraded by trypsin but not by thermolysin (Fig. 3A, row 1). Because Toc75 is deeply embedded in the chloroplast outer envelope membrane, thermolysin could not access the protein. However, because trypsin is able to penetrate the outer membrane, it was able to completely digest Toc75 (as in Fig. 1). In contrast, because neither thermolysin nor trypsin can penetrate the inner envelope membrane, prSS, a stromal marker, was not digested by either protease (Fig. 3A, row 5).
It has previously been reported that when tpToc75-mSS is imported into chloroplasts, the processed product is exposed to the chloroplast intermembrane space in both soluble and membrane-bound forms (18). Thus, we utilized this construct as a marker for the intermembrane space. Accordingly, we found that both the soluble and membrane-bound products generated from tpToc75-mSS were degraded by trypsin but not by thermolysin (Fig. 3A, row 4). If Tic110 was also exposed to the intermembrane space, we would have expected it to have a protease sensitivity similar to tpToc75-mSS. However, neither of the Tic110 constructs, tp110 -110N and tpSS-110N, which were expected to have the same topology as Tic110 itself (23), were degraded by trypsin as long as protease activity was sufficiently quenched by the protease inhibitor mixture (Fig.  3A, rows 2 and 3, compare trypsin I and trypsin II protocols). We interpreted these results to indicate that these Tic110 constructs were not exposed to the intermembrane space. Since it has been previously demonstrated that these two constructs are inserted in the inner envelope membrane (23), we con- 2. Tic110 is degraded by insufficiently quenched trypsin  only after chloroplast lysis. A and B, intact chloroplasts (50 g of  chlorophyll) were incubated for 10 min with trypsin (500 g of protease/mg of chlorophyll). Protease activity was quenched either with a mixture of protease inhibitors (ϩ) or with 1 mM PMSF (Ϫ) at the stages indicated above and as outlined under "Results." Intact chloroplasts were reisolated, lysed, and separated into membrane and soluble protein fractions. Half of each membrane fraction was analyzed by SDS-PAGE and immunoblotting with antiserum against Tic110. cluded that they must be oriented toward the chloroplast stroma.
We also examined the protease sensitivity of three endogenous proteins in these chloroplasts (Fig. 3B). Tic110 was not significantly degraded by either thermolysin or trypsin as long as trypsin was adequately quenched (Fig. 3B, lanes 1-4). This is similar to the results obtained in our previous experiments and those seen for the imported Tic110 constructs. Toc75 was degraded by trypsin but not by thermolysin (Fig. 3B, lanes  5-8), consistent with the results obtained for imported Toc75. On the other hand, Toc34 was degraded by both proteases (Fig.  3B, lanes 9 -12). These results are consistent with the fact that the cytosolic domain of Toc34 is exposed on the outer surface of chloroplasts (7,11).
Tic110 Is Exposed on the Same Face of Inner Envelope Membrane Vesicles as ClpC, a Stroma-facing Protein-The results presented above suggest that within intact chloroplasts, Tic110 is oriented toward the stromal compartment. In order to extend and confirm this conclusion, we analyzed the topology of Tic110 in a second system, isolated inner envelope membrane vesicles. Specifically, we compared the trypsin sensitivity of Tic110 to that of ClpC, a stromal hsp100 homologue. ClpC is primarily a soluble protein; however, it is known that a significant portion of the ClpC molecules in the chloroplast are associated with the stromal side of the inner envelope membrane (28,29). Therefore, if Tic110 is indeed exposed on the stromal face of the inner envelope membrane, it should display the same trypsin sensitivity as ClpC. This indeed was what we observed upon analysis of inner membrane vesicles (Fig. 4). Both Tic110 and ClpC were resistant to degradation at low protease concentrations and susceptible at higher levels of trypsin. In addition, both proteins began to be significantly degraded at the same trypsin concentration (Fig. 4, A, lane 5, and B, lane 5), indicating that Tic110 and ClpC were exposed on the same side of the vesicles. Consequently, we concluded that Tic110, like ClpC, was oriented toward the chloroplast stroma.

DISCUSSION
To investigate the process of protein import into chloroplasts in detail, it will be necessary to study the translocation machineries of the outer and inner envelope membranes separately, as has been done for the mitochondrial protein import system (30). Mitochondria, like chloroplasts, are surrounded by an envelope composed of two separate membranes. Techniques have been developed to physically remove the mitochondrial outer envelope membrane so that inner envelope membrane proteins can be specifically analyzed (31,32). Mitoplasts, mitochondria in which the outer membrane has been selectively ruptured and/or dissolved, can be generated either by subjecting intact mitochondria to osmotic shock treatment (31) or by treating them with digitonin (32). These two methods have been used successfully to study the location and topology of mitochondrial inner envelope membrane proteins and the mechanism of mitochondrial protein import (for example, see Refs. [33][34][35][36][37][38]. Similar techniques to selectively remove the outer membrane of chloroplast envelopes are not yet available. In lieu of such approaches, investigations on chloroplast inner envelope membrane proteins have relied on the ability of certain proteases, specifically trypsin, to selectively destroy the permeability barrier of the outer membrane and degrade inner membrane proteins that are exposed to the intermembrane space while leaving stromally exposed proteins intact (19 -21). This technique can thus be used to differentiate between an intermembrane space and a stromal localization for both soluble and membrane proteins (13,14,18,23), as we have done in this study.
Two independent investigations have provided evidence indicating that Tic110 is a component of the chloroplast protein translocation apparatus localized in the inner envelope membrane (13,14). However, no function for Tic110 during protein translocation has been clearly established. Elucidating the topology of Tic110, about which the original reports disagreed (13,14), will be an important first step toward understanding the role of this protein in the import process. In this investigation, we have provided evidence indicating that the large (Ͼ90-kDa) hydrophilic domain of Tic110 was oriented toward the stromal compartment. Because the one or two predicted transmembrane domains of Tic110 are near the N terminus (within the first 10% of the mature protein), it is likely that the regions of Tic110 that are important for its function reside within the large hydrophilic domain, which we have localized.
Previous investigations have proposed that Tic110 may be involved in mediating the interaction between outer and inner envelope membrane translocation components during protein import (14). However, our evidence does not support this view. The stromal orientation of the major portion of Tic110 would probably not allow this protein to interact with outer envelope membrane proteins. Instead, it is more likely that Tic110 interacts with stromal components of the translocation apparatus. For instance, Tic110 may be involved in the recruitment of molecular chaperones, including ClpC, to the site of protein import.
This study has demonstrated that Tic110 is degraded by trypsin only when trypsin-treated chloroplasts are insufficiently quenched. Incomplete quenching of trypsin activity with PMSF is the most likely explanation for previous reports concluding, based on trypsin analysis, that Tic110 is degraded by the protease and thus is oriented toward the intermembrane space (14). An investigation reported by Kessler and Blobel (13) on the topology of Tic110 also utilized trypsin digestion data to analyze this problem. These investigators, who found that trypsin does not degrade Tic110, quenched protease activity with a combination of inhibitors. Their results support our claim that when trypsin is adequately quenched, Tic110 remains largely undigested after protease treatment of intact chloroplasts and provide further evidence for the conclusion that the large hydrophilic domain of Tic110 is exposed to the chloroplast stroma rather than the intermembrane space.
In the case where trypsin was insufficiently quenched, it is possible that active trypsin either bound to the chloroplast envelope membranes or was trapped in the intermembrane space and, consequently, was retained during reisolation of intact chloroplasts. Then, during or after chloroplast lysis, trypsin was able to gain access to proteins exposed on the stromal face of the inner envelope membrane and digest them. In this investigation, we analyzed the protease sensitivity of newly imported, radiolabeled SS, a stromally localized protein. This protein did not seem to be significantly degraded by trypsin, regardless of the method used to quench protease activity. To explain these observations, we propose that incompletely quenched trypsin is "trapped" inside envelope vesicles that form upon chloroplast lysis and is thus unable to significantly degrade soluble proteins, including SS. During separation of membrane and soluble proteins, the protease would be sedimented with the membrane vesicles away from soluble molecules, including any quenching reagents added during or after the lysis stage. When membrane proteins are subsequently solubilized in SDS-PAGE sample buffer, active trypsin can be released from the vesicles and degrade Tic110 and perhaps other membrane proteins as well. It is thus necessary to adequately quench trypsin either before or at the time of chloroplast lysis in order to prevent active protease from being released after chloroplasts have been ruptured (Fig. 2).
It should be noted that in this investigation we did not completely repeat the results of Lü beck et al. (23). During the trypsin digestion of the newly imported, radiolabeled precursor constructs, we utilized a lower trypsin concentration (500 g of trypsin/mg of chlorophyll versus 1000 g of trypsin/mg of chlorophyll) and a different quenching protocol (protease inhibitor mixture or PMSF versus PMSF and trypsin inhibitor). The protocol we utilized during these import experiments was the same used in all of the other experiments presented in this report. Although we did not completely repeat the protocol of Lü beck et al. (23), we do not believe this significantly affected our results or the conclusions we have drawn from them, since all other observations indicate that Tic110 does indeed face the chloroplast stroma.
During the course of this investigation, we also attempted to specifically label proteins exposed to the intermembrane space with biotinylation reagents that supposedly could not permeate the inner envelope membrane. However, we observed that these reagents were able to enter the chloroplast stroma in small but significant quantities, and we were unable to develop reaction conditions to prevent labeling of stromal proteins.
Thus, it appears that at the current time, trypsin digestion analysis is the most reliable method to analyze the topology of inner envelope membrane proteins. Our experiments have revealed that the protocol used to quench trypsin activity during such studies can have a major effect on the results of experiments and thus the conclusions drawn from these results (Fig.  1). Consequently, it is important that measures be taken to ensure that trypsin is adequately quenched whenever this protease is used as an analytical tool. When our quench mixture is added to intact chloroplasts prior to trypsin addition, Tic110 and Toc75 (an outer envelope membrane protein normally susceptible to trypsin action) are left largely intact (Fig. 1). Thus, we concluded that the protease inhibitor mixture used in this investigation was sufficient to quench trypsin activity. Such a mixture of inhibitors should be useful in studying the topology of membrane proteins or in any other investigations where analysis by trypsin digestion plays a pivotal role.