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Originally published In Press as doi:10.1074/jbc.M401403200 on February 19, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21533-21542, May 14, 2004
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Regulation of Immature Protein Dynamics in the Endoplasmic Reticulum*

Asako Kamada{ddagger}§, Hisao Nagaya§, Taku Tamura§, Masataka Kinjo||, Hai-Ying Jin{ddagger}, Toshiharu Yamashita{ddagger}, Kowichi Jimbow{ddagger}, Hideo Kanoh**, and Ikuo Wada§{ddagger}{ddagger}

From the Departments of {ddagger}Dermatology and **Biochemistry, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan, the §Core Research for Evolutional Science and Technology, JST, Japan, the Department of Cell Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295 Japan, and the ||Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan

Received for publication, February 9, 2004 , and in revised form, February 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The quality of nascent protein folding in vivo is influenced by the microdynamics of the proteins. Excessive collisions between proteins may lead to terminal misfolding, and the frequency of protein interactions with molecular chaperones determines their folding rates. However, it is unclear how immature protein dynamics are regulated. In this study, we analyzed the diffusion of immature tyrosinase in the endoplasmic reticulum (ER) of non-pigmented cells by taking advantage of the thermal sensitivity of the tyrosinase. The diffusion of tyrosinase tagged with yellow fluorescence protein (YFP) in living cells was directly measured using fluorescent correlation spectroscopy. The diffusion of folded tyrosinase in the ER of cells treated with brefeldin A, as measured by fluorescent correlation spectroscopy, was critically affected by the expression level of tyrosinase-YFP. Under defined conditions in which random diffusional motion of folded protein was allowed, we found that the millisecond-order diffusion rate observed for folded tyrosinase almost disappeared for the misfolded molecules synthesized at a nonpermissive high temperature. This was not because of enhanced aggregation at the high temperature, as terminally misfolded tyrosinase synthesized in the absence of calnexin interactions showed comparable, albeit slightly slower, diffusion. Yet, the thermally misfolded tyrosinase was not immobilized when measured by fluorescence recovery after photobleaching. In contrast, terminally misfolded tyrosinase synthesized in cells in which {alpha}-glucosidases were inhibited showed extensive immobilization. Hence, we suggest that the ER represses random fluctuations of immature tyrosinase molecules while preventing their immobilization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The maturation of proteins in the secretory pathway requires various sequential reactions including suppression of backward movements through the translocational channel by BiP (1); cleavage of the signal sequence and attachment of N-linked oligosaccharides (2); the prevention of nonproductive folding intermediates by various molecular chaperones (3); disulfide shuffling by disulfide isomerases possessing chaperone activity (4, 5); and proline isomerization (6). When these processes are not completed, proteins are disposed eventually. Recent extensive studies have been revealing the underlying molecular mechanisms of this destruction (7-11). However, there are also misfolded proteins that are able to maintain reversible folding for several hours after synthesis but somehow fail to exit the ER.1A temperature-sensitive ts045 variant of the vesicular stomatitis virus glycoprotein (VSVG) is the best known example (12-14). It was thought that the defect in the exit of this glycoprotein from the ER and its transport to the Golgi could be explained by (a) uncompleted interactions of cargo proteins with the "ER matrix," which is composed of various chaperones and folding enzymes (15, 16) or (b) misfolded aggregates (13) that are too large to enter the COPII-coated ER exit sites. However, measurements of the misfolded VSVG mutant in living cells using fluorescence recovery after photobleaching (FRAP) (17) surprisingly showed that the thermal-induced misfolding caused no significant loss of lateral mobility (18). While various models can be conceived to explain the results, the study at least eliminated the possibility that the misfolded VSVG failed to exit because it is tethered to the immobile ER matrix (19).

Movements of newly synthesized proteins could affect their folding per se. Collision of two molecules exposing hydrophobic patches on their surfaces could result in the formation of aggregates. Also, folding rates depend on the frequency of collision with molecular chaperones. Hence, it is expected that the mobility of proteins during folding is regulated in living cells. However, this is hard to measure by FRAP because FRAP records the average motion of a mass population and its time resolution is not sufficient for complicated dynamics including submillisecond diffusion. For measuring such random diffusional motion of an individual protein, fluorescence correlation spectroscopy (FCS) is, at present, the only practical method (20-22). FCS detects fluctuation of the fluorescence intensity in a confocally defined volume with a sharply focused laser. This method has been developed as a unique technique to measure translational and rotational diffusion coefficients of molecules in solution and in living cells (22-25). Application of this technique to biological systems has brought to light novel aspects of various molecular dynamisms, such as the status of the tubulin complex in kinesin-mediated axonal transport in the squid giant axon (26). However, this method has not been used to analyze the process of protein maturation in living cells.

To study the dynamics of immature proteins, we reasoned that we might be able to use the thermal sensitivity of tyrosinase, a melanosomal membrane enzyme catalyzing the oxidation of monohydric phenols, a critical step in melanin biosynthesis (EC 1.14.18.1 [EC] ) (27). Failure to express its catalytic activity results in the occurrence of oculocutaneous albinism type I (28). Pigment cells from individuals with some types of this disorder are temperature-sensitive (29, 30). When wild type tyrosinase is expressed in non-pigmented cells, its folding is also sensitive to heat (31). Completion of tyrosinase folding is a well defined step in that it can be monitored by the acquisition of dihydroxyphenylalanine (DOPA) oxidizing activity either in situ or in solution, and it depends entirely on interactions with calnexin (32-35). In non-pigmented cells, tyrosinase is transported to endosomal/lysosomal vesicles (36-40). In this study we first tested whether the diffusion of folded tyrosinase is measurable. We then studied how the dynamics change in the thermally restricted condition. We also used FRAP to determine the fraction of the population that was immobilized. Our results show that folding appears to be regulated at various levels of microdynamics.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Vectors and Culture Cells—The adenovirus vector used to express tyrosinase is described elsewhere (41). EcoRI and BamHI sites were attached to the human tyrosinase cDNA (35) by polymerase chain reaction (PCR). Tyrosinase fused to yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) was then produced by ligating the human tyrosinase into pEYFP-N1 or pECFP-N1 (BD Bioscience) cut with EcoRI and BamHI, respectively. For construction of the Sar1(T39N) expression vector, Sar1 cDNA was isolated by reverse transcription from HepG2 RNA using the primer 5'-GGATCAGTCCAGAGAAGTAAAAC-3', then amplified by PCR with the pair of primers 5'-GCCGGAGAGCCCCTCAGGCCGTAGTAAGC-3' and 5'-TCACCGTCCAAACATCAGTCAATATACTGG-3'. Restriction sites for BglII and KpnI were created at the 5' and 3' ends of the Sar1 cDNA using PCR. The Sar1 cDNA was then ligated into the corresponding sites of pEYFP-N1{Delta} whose EYFP open reading frame was removed by restriction with BamHI and NotI followed by ligation after polishing the terminus. To construct the GDP-restricted form of Sar1, the codon for Thr at amino acid 36 was mutated to that encoding Asn using the QuikChangeTM protocol (Stratagene, La Jolla, CA). An expression vector for CFP-GT-(1-81) (GT-(1-81); the amino-terminal 81 amino acids of human {beta}-1,4-galactosyltransferase) were purchased from Clontech. A vector for VSVG (ts045) was described previously (42). These vectors were introduced directly into cells using siliconized glass microbeads (42). SiHa and COS7 cells were obtained from the American Type Culture Collection (Manassas, VA). COS7 cells stably expressing hSec13-YFP were generated as follows. The coding sequence of human Sec13-YFP (42) was subcloned into the appropriate cloning sites of the retrovirus expression vector, pCX4bsr. pCX4bsr, a generous gift from Dr. T. Akagi (Osaka BioScience Institute), is a modified version of pCXbsr (43) that lacks the internal initiation codons within the gag region. The recombinant retrovirus was generated as described (43) and used to infect COS7 cells. The cells expressing hSec13 were selected by culturing them in Dulbecco's modified Eagle's medium containing blasticidin (10 µg/ml).

Analysis of Protein Dynamics in Living Cells—Live cell analysis using the microscopy was essentially described previously (42). The culture temperature of cells on the microscope stage was controlled using an objective heater for a planapochromat lens x63 (Bioptechs, Butler, PA) or a Silicon Heater (Cell MicroControls, Norfolk, VA) for a C-Apochromat x40 lens in combination with a stage heater (Kitazato Supply, Fujinomiya, Japan). To monitor the temperature of the cells under observation, an infrared thermometer (model CT820, CITIZEN Co., Tokyo, Japan) was used. For confocal microscopy and FCS analysis, a ConfoCor2 instrument (Zeiss, Jena, Germany) was used. Confocal images were taken with the laser scanning microscopy module. The excitation light of an argon ion laser at 514 nm was reflected by a dichroic mirror (HFT 514) and focused through a C-Apochromat x40, NA = 1.2 water immersion objective (pinhole width 70 µm). A 530-560 nm band-pass filter was used to filter out the remaining scattered laser light. In all measurements, the minimum laser power of the setup was used. The fluorescence signal was recorded for three consecutive periods of 15 s (time resolution, 200 ns). The autocorrelation function and data fitting was performed with the software provided with the setup. Indirect immunofluorescence of fixed cells and time-lapse analysis of fluorescent molecules in live cells were carried out and processed as described previously by Nagaya et al. (42). For FRAP experiments, an area (1 µm2) was exposed to the maximum power of the argon laser and then the recovery from the bleaching was measured at the minimum power of the laser. The obtained data were analyzed by fitting to a formula for one-dimensional diffusion (44) using Prism version 3.0 software (San Diego, CA).

Tyrosinase Activity Measurements and Sedimentation Analysis—Active staining of DOPA oxidase and spectrophotomeric measurements of DOPA oxidase activity in cellular lysates were carried out as described (35). To determine the sedimentation velocity of tyrosinase synthesized under various culture conditions, cells in a 60-mm dish were lysed with 0.5 ml of 1% sodium cholate, 0.15 M NaCl, 20 mM potassium phosphate, pH 7.2, and directly loaded onto a sucrose gradient from 25 to 10% in 0.3% sodium cholate, 0.15 M NaCl, 20 mM potassium phosphate, pH 7.2, in a tube for a Hitachi RPS40T rotor. After centrifugation at 36,000 rpm for 18 h at 4 °C, ten 1-ml fractions were collected from the bottom of the tube and 10 µl from each fraction was subjected to immunoblot analysis using an anti-tyrosinase monoclonal antibody (Novocastra Laboratories Ltd., Balliol Business Park West, UK).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FCS Measurements of Tyrosinase Expressed in Non-pigmented Cells—To test whether we could use the reported thermal sensitivity of tyrosinase folding to synchronize its maturation, we first examined the heat lability of its folding and stability. A recombinant adenovirus was used to express tyrosinase in human cervical carcinoma SiHa cells for 24 h at 37 °C and at various other temperatures (Fig. 1A). For the cells incubated at 37 °C, protein synthesis was halted by the addition of puromycin and the cells were further incubated for 24 h at the indicated temperatures. The cells were then lysed with detergents and the DOPA oxidase activity was determined (35). The DOPA oxidase activity was dramatically reduced when tyrosinase was synthesized above 39 °C (Fig. 1A, inset). In contrast, thermal treatment of folded tyrosinase (i.e. tyrosinase synthesized at 37 °C) had little effects on the activity, indicating that completion of tyrosinase folding is arrested above 39 °C. Importantly, when the inactive tyrosinase formed at 40 °C was further incubated at 37 °C in the presence of puromycin, the protein became almost fully active with 60 min (Fig. 1A, squares). This activation was not observed in ATP-depleted medium (circles) or in the presence of an {alpha}-glucosidase inhibitor (triangles).



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  		FIG. 1.
Determination of conditions for measuring the diffusional mobility of tyrosinase using FCS. A, thermal synchronization of tyrosinase folding. SiHa cells infected with a recombinant adenovirus expressing wild type tyrosinase were incubated at 40 °C for 24 h and then incubated at 37 °C for the indicated periods in the presence of 40 µM puromycin. Squares, complete medium; circles, ATP-depleted medium; triangles, medium with an {alpha}-glucosidase inhibitor, castanospermine. Inset, shaded bars, sensitivity of folding to the culture temperature. Tyrosinase was expressed at the indicated temperatures for 24 h. Black bars, stability of mature tyrosinase. Tyrosinase was expressed at 37 °C for 24 h, then incubated at the indicated temperature with 40 µM puromycin for 24 h. The cells were lysed, then the DOPA oxidase activity of the lysates was measured. The amounts of tyrosinase in the lysates were determined by immunoblots. The enzyme activity was expressed as relative activity per immunoreactive tyrosinase. The average of three experiments is shown. B, fluorescence intensity (count rate) of tyrosinase-YFP before (right) and after (left) fixation. An expression vector for tyrosinase-YFP was used to coat beads that were then loaded into COS7 cells and incubated in the presence of brefeldin A (5 µg/ml) for 2 h at 37 °C. The fluorescent signal in a spot in the ER was then counted for 45 s in the living cells (right) or recorded in the same medium after fixation with 3.7% formalin for 10 min at 37 °C (left). C, photobleaching is affected by expression levels. Tyrosinase-YFP was expressed as in B and the fluorescence signal of spots in cells was measured. The degree of photobleaching for 45 s was calculated according to the equation: (Iini - Imin)/Iini, where Iini is the initial rate and Imin the minimum rate during a 45-s recording period (x, living cells; dots, fixed cells). Assuming that the relationship fits with the saturation binding curve, the best-fitting curve was determined using PRISM software. The area of 95% confidence is indicated by the dotted lines.

 
We then used this protocol to study how the mobility of tyrosinase is regulated during maturation. We reasoned that the diffusion profile of thermally misfolded tyrosinase may be distinct from the properly folded molecule if there is any regulation of mobility. To analyze mobility, we used FCS because this technique has the highest time resolution of the available techniques. However, it is known that FCS is often too sensitive to analyze cellular processes (21). In particular, photobleaching is a major obstacle. We therefore examined the conditions in which FCS could detect random diffusion in living cells. Because the degree of photobleaching obtained by FCS should correlate with the density of the molecules, we investigated which expression level would largely allow for random diffusion. In cells where the expression level is too high, the molecules would be at least partially immobilized. To achieve various levels of expression, we used siliconized glass microbeads (42). When photon-counting of tyrosinase-YFP was carried out in formalin-fixed cells, all fluorophores showed rapid decay of fluorescence to near background within 45 s, as expected (Fig. 1B, left panel). The fluctuation of the signal was almost at the level of noise, and no autocorrelation was observed from this counting (not shown). We next examined whether this setup could detect diffusion of tyrosinase-YFP expressed in a living cell at 37 °C for 2 h in the presence of brefeldin A, which prevents export from the ER (45). In this measurement, the degree of photobleaching was reduced compared with that in the fixed cells, and massive signal fluctuation was observed (Fig. 1B, right panel).

When we plotted the extent of bleaching, B, against the expression level, Iini, we noticed that the relationship largely fits a simple saturable binding model: B = Bmax·Iini/(K + Iini). The constant K reflects the degree of correlation between photobleaching and the expression level. In the case of fixed cells, K should be ~0. If the level of tyrosinase expression has little influence on diffusional motion in the membrane, K should far exceed Iini. This would happen if the fluorophore is very bright, so that the number of expressed molecules is very small. However, when measured in the living cells, this is not the case. In the living cells, a significant correlation was observed between the two values when the initial count rate was below 100 kHz (Fig. 1C, x), suggesting that at least tyrosinase-YFP was not saturated at this range and, thus, random diffusion of tyrosinase-YFP should be measurable.

Regulation of Diffusional Mobility of Thermally Misfolded Tyrosinase—We next expressed tyrosinase-YFP at 40 °C and plotted the degree of photobleaching against the number of misfolded molecules. Surprisingly, massive photobleaching was observed irrespective of the expression level (Fig. 2A). A typical decay curve is shown in Fig. 2B (top panel), which resembles that of the fixed protein (Fig. 1B). It should be noted that, different from the fixed cells, the decay in living cells reached a plateau at a significantly higher level than background (~1 kHz), suggesting the presence of a slow movement of nonbleached tyrosinase into the bleached area. However, this mobility was too slow to be captured by FCS. Nonetheless, the counting showed almost no autocorrelation over time (Fig. 2C, black line). To know if this lack of fluctuation is reversed when folding is allowed, we recorded the fluorescence in the same spot over a temperature shift from 40 °C (Fig. 2B, top) to 37 °C (Fig. 2B, middle and bottom). In these cases, fluctuation emerged upon temperature shift (Fig. 2B, middle). Interestingly, an almost identical autocorrelation curve was observed when incubation was continued at 37 °C for another 10 min (Fig. 2C, red line), except for a slight increase in the population with the faster mobility in the millisecond range. A two-component fitting analysis indicated that the mobility was composed of fast (T = 0.64 ms, 50.7%) and slow (T = 9.17 ms, 49.3%) diffusion components (Fig. 2C, bottom panel).



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  		FIG. 2.
FCS measurements of thermally misfolded tyrosinase. Tyrosinase-YFP was expressed at 40 °C for 2 h and its diffusional motion in the ER was measured by FCS. A, photobleaching of thermally misfolded tyrosinase. The photobleached fraction was plotted against the expression levels as in Fig. 1C. In this case, the photobleached fraction does not depend on the initial intensity. B, changes in fluorescence fluctuation upon temperature shift. Immediately after synthesis at 40 °C for 2 h, fluorescence was recorded in the ER for 45 s (top). The same cells were then incubated on the microscopic stage at 37 °C in the presence of 40 µM puromycin and the fluorescence of the same spot was recorded at 10 (middle) and 20 min (bottom). C, autocorrelation function of B. Autocorrelation function of the last 15 s of the recording period is shown at 0 (top panel, black line), 10 (green line), and 20 min (black line) after the temperature shift to 37 °C. Thermally misfolded tyrosinase showed little autocorrelation (top panel, black line). The best-fit two-component simulation profile (T = 0.64 ms, 50.7% and T = 9.16 ms, 49.3%) of the obtained autocorrelation function data at 20 min is shown in the bottom panel (black line).

 
While these results indicated that the random diffusion detected by FCS was suppressed at 40 °C, they also suggested that the protein was slowly mobile at this temperature. We therefore examined whether such dynamics might be detected by FRAP. To test this possibility, we first assayed the rates of reversible photobleaching in fixed cells (46). An area of a formalin-fixed tyrosinase-YFP was photobleached by a pulse of intense laser beam, and the recovery rate was determined. As shown in Fig. 3A, the maximum laser beam reduced the signal to 42% of the initial level and within 1 s the intensity increased to 50% and remained there, suggesting that any recovery above 50% should be because of fluorophore exchange between the bleached and unbleached areas. We then determined the fraction of immobilized molecules in the living cells. While the fluorescence intensity of an area of tyrosinase-YFP synthesized for 2 h at 40 °C was reduced almost as much as it was in the fixed cell by an intense laser beam, the signal in the area recovered almost fully (Fig. 3B, red). Similarly, full recovery from photobleaching was observed in the adjacent area when the cell was incubated on the stage at 37 °C for 15 min after 40 °C expression for 2 h although it appeared to be slightly faster (Fig. 3B, blue). These results and further independent experiments, as summarized in Fig. 3C, indicates that tyrosinase synthesized at 40 °C was slow, but fully mobile.



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  		FIG. 3.
FRAP analysis. Tyrosinase-YFP expression vector was bead loaded onto cells and expressed for 2 h at 40 °C. A, FRAP analysis was performed in a formalin-fixed cell. Images of the cell before and after FRAP are shown. The arrow indicates a photobleached region. B, FRAP analysis of a living cell. Recovery rates after photobleaching were measured in an adjacent area of the same single cell at 0 (red) or 15 min (blue) after a temperature shift to 37 °C in the presence of puromycin. C, summary of maximum recovery rates. FRAP was performed in only one region per cell to minimize loss of the mobile fraction by another round of FRAP. n, number of measurements.

 
Diffusional Mobility of Aggregated Tyrosinase Formed in Castanospermine-treated Cells—To know whether the apparent lack of random diffusion seen with FCS measurements at non-permissive temperatures was because of the formation of large aggregates, we measured the molecular weight of detergent-solubilized tyrosinase using sedimentation analysis through sucrose density gradients. As shown in Fig. 4A, tyrosinase synthesized at the non-permissive temperature, 40 °C, was mostly found in the bottom three fractions of the gradient (red line). After 30 min at 37 °C, the majority was still found in the bottom fractions, but a slight increase in the recovery in fractions 4 and 5 was observed (green line). After 1 h at 37 °C, a small decrease in the percentage of tyrosinase in the bottom fraction was detected (blue line). An almost identical pattern was obtained at 2 h postincubation.2However, the molecules did not acquire the compact tertiary structure detected for the native enzyme synthesized without exposure to 40 °C (black line). When we examined terminally misfolded tyrosinase synthesized in the presence of an {alpha}-glucosidase inhibitor, castanospermine, the majority of the molecules were recovered in the bottom fractions, suggesting that they are largely aggregated. This analysis suggests that a slight alteration in size of tyrosinase aggregates is associated with the acquisition of activity, and that the thermally misfolded tyrosinase is structurally distinct from terminally misfolded tyrosinase.



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  		FIG. 4.
Characterization of tyrosinase maturation. A, sedimentation velocity analysis of tyrosinase. Tyrosinase was expressed in SiHa cells at 37 (black box) or 40 °C (other symbols) for 24 h in the presence (gray box) or absence (other symbols) of 1 mM castanospermine, and then incubated at 37 °C with puromycin. Cells were lysed and applied to a sucrose gradient of 10-25%. The gradients were centrifuged and the fractions were collected from the bottom of the tube. As molecular mass markers, bovine serum albumin (5 S), catalase (11 S), and apoferritin (17 S) were recovered in fractions 8, 5-7, and 4-5, respectively. See "Experimental Procedures" for details. B, active staining of tyrosinase in situ at the non-permissive or the permissive temperature. Tyrosinase was expressed in SiHa cells at 37 (left panels) or 40 °C (right panels) for 24 h using recombinant adenovirus, and stained with anti-tyrosinase antibody (top panels) followed by an Alexa 488-labeled anti-mouse antibody or DOPA staining in situ (bottom panels) as described (35). Fluorescence (top panels) and transmission images (bottom panels) are shown. C, GDP-restricted form of Sar1 inhibits ER export of tyrosinase. The Sar1(T39N) expression vector was co-loaded into COS7 cells with the tyrosinase-YFP expression vector using glass beads. Tyrosinase-YFP fluorescence observed at 3 h post-loading was directly recorded and the active staining pattern was recorded as a transmission image.

 

To examine the possibility that the suppression of millisecond diffusion at the non-permissive temperature was caused by enhanced aggregation, we measured diffusion of the terminally misfolded tyrosinase synthesized in castanospermine-treated cells at 37 °C. However, in this case, the autocorrelation analysis showed that the diffusion of the slower mobility component was slightly slower but detectable by FCS (Fig. 5A). The typical measurement showed that the diffusion was composed of a fast (586.8 µs, 45.6%) and a slow (18.7 ms, 54.4%) diffusion (Fig. 5A). A summary of FCS measurements at various conditions is also shown in Fig. 5B. Hence, we conclude that the lack of autocorrelation at the non-permissive temperature was not the result of enlarged aggregates. However, FRAP analysis of the castanospermine-treated cells showed that the maximum recovery rate was reduced by ~30% (Fig. 5C). Taken together, we reasoned that long range diffusion, which is not observed in triglucosylated tyrosinase, is directly associated with the acquisition of the enzyme activity.



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  		FIG. 5.
FCS and FRAP analysis of tyrosinase-YFP in castanospermine-treated cells. Misfolded tyrosinase-YFP expressed in the presence of castanospermine at 37 °C for 2 h was subjected to FCS (A) or FRAP (C) analysis as described in the legends to Figs. 2 and 3, respectively. Panel A, best fitting two-component simulation profile (T = 0.59 ms, 45.6%, and T = 18.7 ms, 54.4%) (black line) and the autocorrelation function data (blue line). Inset, fluorescence fluctuation (the last 15 s of the recording period of three consecutive recordings) used for the autocorrelation analysis. Panel B, summary of FCS measurements. In the upper panels, fast and slow diffusion time in cells treated as indicated are plotted against the percentage of subpopulation estimated as in panel A. Averaged values and S.E. at each condition were calculated and are shown in the table. N.D., not determined because of lack of autocorrelation. Panel C, recovery rates after photobleaching. The error bars are the measured S.E. (n = 6).

 
Enzymatically Active but Non-native Tyrosinase Was Transported to Lysosomes—Finally, we studied the fate of tyrosinase after a temperature shift to investigate whether the non-native but fully diffusible tyrosinase is properly targeted to lysosomes. The thermally misfolded tyrosinase was confined in the ER as expected (Fig. 4B). When a GDP-restricted form of Sar1, whose expression inhibits export of cargo from the COPII-coated ER exit sites, was co-expressed with tyrosinase at 37 °C, tyrosinase was retained in the ER and DOPA oxidase activity was expressed (Fig. 4C). Indeed, at 4 min after the temperature shift, at least some tyrosinase appeared to be concentrated in hSec13-coated structures (Fig. 6A). As shown in Fig. 7, time-lapse analysis revealed that transport to the Golgi apparatus, which was demarcated by coexpression of the Golgi marker galactosyltransferase-(1-81)-CFP, was clearly observed at 30 min after a temperature shift from 40 to 37 °C (Fig. 7, red). Further incubation allowed the initial formation of small vesicles at 50 min and of large granules at 72 min. The larger granules were identified as lysosomes because tyrosinase colocalized with lgp85 within them.2Interestingly, tyrosinase was found in compartments adjacent to lysosomes before merging with pepstatin-positive lysosomes (Fig. 6B, bottom). Thus, acquisition of enzyme activity and proper targeting to lysosomes in a classical pathway appears to occur only when diffusion in the millisecond range and slow but full exchange in the ER are allowed during maturation in the ER.



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  		FIG. 6.
Targeting of tyrosinase-CFP in COS7 cells. A, tyrosinase-CFP was concentrated in COPII-coated structures. Tyrosinase-CFP was expressed at 40 °C for 2 h in COS7 cells stably expressing hSec13-YFP. After the addition of puromycin, the cells were placed on a microscope stage and incubated at 37 °C. Shown are images in the ER network at 4 min. Most punctate structures containing tyrosinase-CFP (red) were coated with hSec13-YFP (green, white arrows). B, targeting of tyrosinase to lysosomes. Tyrosinase-CFP (red) was expressed as in A and incubated at 20 °C for 2 h in the presence of BODYPY-FL-labeled pepstatin (25 ng/ml, green) and puromycin. The medium was then changed to fresh medium without the labeled pepstatin and incubated for 1 or 2 h at 37 °C. At 1 h, most tyrosinase-containing structures were found in structures adjacent to pepstatin-positive vesicles, as shown in the enlarged images in the 60-min column. After incubation for another hour, most of the tyrosinase signals became completely merged with the fluorescent pepstatin signals, as shown in the enlarged images in the 120-min column.

 



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  		FIG. 7.
Transport of tyrosinase to lysosomes in a single cell. Expression vectors for tyrosinase-YFP and CFP-GT-(1-81) were introduced into COS7 cells by siliconized glass microbeads and incubated for 2 h at 40 °C. The temperature was lowered to 37 °C after the addition of 40 µM puromycin and then both fluorescent images were recorded at 20-s intervals. The green images represent tyrosinase-YFP and the red images represent CFP-GT-(1-81) in the same cell.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The dynamics of proteins in living cells are regulated at various levels. Folding depends on the molecules' "breathing," that is, on fluctuations in the relative positions of amino acid residues, but excessive collisions may lead to nonproductive interactions. We therefore reasoned that diffusion should be regulated upon maturation. To examine this process in living cells, it is necessary to manipulate the status of folding. The ts045 strain of VSVG (47) was the only well characterized cargo available for this purpose. In this paper, we took advantage of the thermal sensitivity of tyrosinase to explore the possibility that tyrosinase folding can be manipulated. We initially examined various naturally occurring temperature-sensitive tyrosinase mutants such as R402Q, P406L, and R422Q (30). However, their thermally induced misfolding was not very reversible and therefore not suitable for experiments to study maturation.2However, the folding of wild type tyrosinase was dependent on the incubation temperature, as we were able to show using DOPA oxidase activity as a folding marker (Fig. 1A). Although incubation at the permissive temperature did not resolve the structure to the completely native structure (Fig. 4A), the molecule still acquired COPII-dependant exportability from the ER (Figs. 4C and 6A) as well as full DOPA oxidase activity (Fig. 1A). We reasoned that these properties were adequate to study the dynamic properties of a maturing cargo protein in the ER.

At present, the expression of chimeric proteins tagged with a fluorescent protein is the only way to study diffusion in living cells. We noticed that the conventional methods for transiently expressing tyrosinase-YFP using lipophilic compounds generally resulted in massive photobleaching when measured with a FCS setup. As a result, we first determined how expression levels affect photobleaching using a glass bead-loading method (42) (Fig. 1C). The relationship suggests that any detectable levels of folded tyrosinase-YFP expression significantly influence diffusion in the ER, but as long as the expression level is below a threshold, random diffusion should be measurable. We estimated that tyrosinase-YFP would not be "saturated" in our setup if the expression level was less than 100 kHz. If we assume that the diameter of folded tyrosinase is smaller than 10 nm and if the fluorescence count per single molecule is higher than 3 kHz, as observed, this estimation seems reasonable because the confocal volume (~0.2 fl), which is estimated to contain fewer than 33 molecules, is far larger than the volume that tyrosinase-YFP occupies. Our first conclusion was that the observed diffusion was markedly suppressed at the non-permissive temperature, as measured with FCS. This is based on the results that thermally misfolded tyrosinase showed extensive photobleaching irrespective of the expression level (Fig. 2A). As a result of the limited fluctuation, the autocorrelation function could not be applied to this measurement (Fig. 2B, top panel, and C). It is conceivable that this result was caused by the formation of extremely large aggregates. However, this is unlikely because the apparent molecular size of the thermally misfolded tyrosinase was no larger than that synthesized in the presence of castanospermine (Fig. 4A).

We thus conjectured that these data indicate a status where random diffusion is restricted by the cellular machinery. This regulation would presumably help to prevent irreversible misfolding because of large aggregate formation by reducing the chance of collision between proteins with exposed hydrophobic patches on their surfaces, and would thereby function to maintain foldability in stressful conditions. Various responses to stress include suppression of translation, induction of heat-shock proteins, and enhanced degradation of misfolded proteins (48). In general, it is thought that aggregation is prevented by the repeated binding of molecular chaperones. Our conclusion may indicate that there is another cellular mechanism that is used to avoid the formation of aggregates, which are thought to be toxic (49-51). Indeed, thermally misfolded tyrosinase showed no particular cytotoxicity, in that it was possible to obtain COS7 cells stably expressing misfolded tyrosinase by culturing them at 40 °C.2This may be partly because of the proposed restriction of random diffusion to prevent the formation of aggregates. The observed slow diffusion could be caused by either a regulated association with the immobile matrix or the association with a matrix whose diffusion is thermally regulated. One candidate for the matrix is the ER chaperone network, which is composed of weakly interacting molecular chaperones and folding enzymes (15, 16). Alternatively, it is possible that cells contain protective structures whose phase or conformation is altered upon heat shock.

Similar restriction of diffusion has been described in several reports. For example, in an analysis of protein transport in the plastid tubules of the tobacco plant, two-photon FCS revealed that spontaneous diffusion of expressed green fluorescent proteins in plastids was ~50 times slower than that in the cytosol (52). Interestingly, they found that the FCS recordings included the presence of ATP-dependent active transport, which alternated with "dim periods" in which only random diffusion was seen. This active transport could function to provide long-range transport in a 50-µm plastid tubule. They suggested that the low diffusion coefficient may be caused by fluid-phase viscosity. Similarly, mitochondrial matrix proteins are thought to have slow diffusion because of steric hindrance because of a very high protein concentration in the compartments (53). Interestingly, the dynamics of proteins in the matrix is rather anomalous. Partikian et al. (54) showed that green fluorescent protein expressed in the matrix was as highly mobile as in water, although large enzyme complexes in the matrix were almost immobilized, suggesting that the mitochondrial matrix is organized into a highly viscous peripheral area and a central region with low protein density. Another well known example is the nuclear proteins. Extensive FRAP analysis has revealed that molecules in the nucleus show highly diverse dynamics (reviewed by Refs. 55-59) that most likely depend on their associations with DNA, which is nearly immobile on a time scale of several minutes (60).

We thus think that the regulation of immature tyrosinase mobility occurs on at least two different levels. At present, our preferred model is that the FRAP-detectable diffusion may be a result of the cellular machinery for facilitated diffusion in the ER network. Considering that immature proteins are transported through the narrow hollow tubules of the ER to the punctate COPII-coated sites, and that the diameter of the ER tubules are only a few times larger than those of average mature proteins, it is not surprising that active transport of molecules in the ER is required for efficient maturation. Indeed, our current research indicates that the mobility of certain proteins in the ER is regulated.3Further studies on the dynamics of maturing proteins should help to understand why folding is so successful in healthy cells continuously exposed to various types of folding stresses that easily terminate folding in vitro.


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (to I. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Cell Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295 Japan. Tel.: 81-24-547-1663; Fax: 81-24-549-8898; E-mail: iwada{at}fmu.ac.jp.

1 The abbreviations used are: ER, endoplasmic reticulum; CFP, cyan fluorescent protein; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; YFP, yellow fluorescent protein; DOPA, dihydroxyphenylalanine; VSVG, vesicular stomatitis virus glycoprotein. Back

2 A. Kamada, H. Nagaya, T. Tamura, M. Kinjo, H.-Y. Jin, T. Yamashita, K. Jimbow, H. Kanoh, and I. Wada, unpublished data. Back

3 H. Nagaya, T. Tamura, K. Hatsuzawa, H. Hashimoto, and I. Wada, manuscript in preparation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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