and of an in lmport system' - plant physiology · plant physiol. (1 995) 107: 1201-1 208...

Plant Physiol. (1 995) 107: 1201-1 208

Development and Application of an in Vivo Plant Peroxisome lmport System'

Adebiyi Banjoko and Richard N. Trelease*

Arizona State University, Department of Botany, Box 871 601, Tempe, Arizona 85287-1 601

l h e purposes of this study are to develop an in vivo cell system that is suitable for the immunofluorescent detection of transiently expressed proteins targeted to plant peroxisomes and to determine whether a C-terminal serine-lysine-leucine (SKL) tripeptide, a consensus-targeting signal for mammalian peroxisomes, also targets proteins to plant peroxisomes. Protoplasts from mesophyll cells and from suspension-cultured cells initially were examined for their potential as an in vivo import system. Severa1 were found suitable, but based on a combination of criteria, suspension-cultured tobacco (Nicofiana fabacum L. cv Bright Yellow 2) cells (TBY-2) were cho- sen. The tobacco cell extracts had catalase activity, and two polypeptides of approximately 55 and 57 kD specifically were detected on immunoblots with anti-cottonseed catalase immunoglobulins C as the probe. lndirect immunofluorescence microscopy with these immunoglobulins C revealed a punctate labeling pattern indicative of endogenous catalase localization within putative TBY-2 peroxisomes. The cells did not have to be completely converted to protoplasts for optimal microscopy; treatment with 0.1 YO (w/v) pectolyase for 2 h was sufficient. Microprojectile bombardment proved superior for transient transformation of the TBY-2 cells with plasmids encoding /3-glucuronidase, or chloramphenicol acetyltransferase (CAT), or CAT with an added C-terminal tripeptide (CAT-SKL). C-terminal SKL i s a consensus, type 1, peroxisome targeting signal. Double indirect immunofluorescent labeling showed that CAT-SKL co-localized with endogenous catalase. Non- punctate, diffuse localization of CAT without SKL provided direct evidence that the C-terminal SKL tripeptide was necessary and sufficient for targeting of CAT to plant peroxisomes. These data demonstrate the effectiveness of this peroxisome targeting signal for plant cells.

Peroxisomes are single membrane-bound organelles present in virtually a11 eukaryotic cells. They possess di- verse sets of enzymes that vary with cell/tissue type and environmental influences. For example, specialized peroxisomes named glyoxysomes possess enzymes for operation of the glyoxylate cycle and for p-oxidation of fatty acids, which are essential pathways for postgerminative growth of oil-rich seedlings (Beevers, 1979; Huang et al., 1983). Peroxisomes do not possess DNA or protein-synthesizing machinery and acquire proteins by import from the cy- tosol, usually without proteolytic processing (Kindl, 1982;

Lazarow and Fujiki, 1985; Borst, 1989; de Hoop and Ab, 1992; van den Bosch et al., 1992).

Primary amino acid sequences act as PTSs for nonproc- essed matrix proteins. Elucidation of necessary and/or sufficient PTSs for import into animal or funga1 peroxisomes have come mostly from in vivo import studies of cultured mammalian (Gould et al., 1989; Swinkels et al., 1992) and yeast (Aitchison et al., 1992; Diste1 et al., 1992; Zhang et al., 1993; Erdman, 1994) cells and from in vitro experiments with isolated rat liver peroxisomes (Miyazawa et al., 1989; Miura et al., 1992). A C-terminal tripeptide, the so-called SKL motif or PTSl type of signal, seems to be the most common PTS (Subramani, 1993), although a cleaved N-terminal sequence (PTS2 type) serves as a signal for rat liver peroxisomal 3-ketoacyl-COA thiolase (Osumi et al., 1991; Swinkels et al., 1991) and watermelon glyoxysomal malate dehydrogenase (Gietl et al., 1994).

Transgenic plants have been used to assess in vivo import of several proteins into plant peroxisomes. Firefly luciferase, a peroxisomal enzyme with a C-terminal SKL, was imported into leaf peroxisomes of transgenic tobacco (Nicotiunu tubacum) plants as well as into peroxisomes in yeast and cultured mammalian cells (Gould et al., 1987, 1990a). The six C-terminal amino acids of spinach glycolate oxidase were sufficient to direct a bacterial protein, GUS, into tobacco leaf peroxisomes (Volikita, 1991). Castor bean ICL was detected in tobacco leaf peroxisomes (Onyeocha et al., 1993), and information contained within the C-terminal 37 amino acids of oilseed rape ICL was necessary for import into leaf and root peroxisomes of Arabidopsis (Olsen et al., 1993). The latter two studies suggest that the protein import machinery is the same among varied types of plant peroxisomes.

The focus of this research was to develop and implement an in vivo, plant peroxisome import system capable of reliably detecting transiently expressed proteins via immunofluorescent microscopy. We evaluated protoplasts prepared from sterilely grown plant explants and suspension-cultured cells derived from several species. Although several of these cell systems seemed suitable for peroxisome import, TBY-2 suspension-cultured cells, treated only with pectolyase, proved to be the most desirable for exper-

This research was supported by National Science Foundation (NSF) grant MCB-930395 to R.N.T. Assistantship support for A.B. was from the Arizona State University Graduate College and NSF grant MCB-930395.

* Corresponding author; e-mail atrntQasuvm.inre.asu.edu; fax 1-602-965- 6899. damine B isothiocyanate.

Abbreviations: BODIPY, N'4odoacetylethylenediamine; CAT, chloramphenicol acetyltransferase; FITC, fluorescein isothiocyanate; ICL, isocitrate lyase; PCV, packed cell volume; PTS, peroxisome targeting signal; SKL, serine-lysine-leucine; TBY-2, tobacco Bright Yellow 2 suspension-cultured cells; TRITC, tetramethylrho-

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1202 Banjoko and Trelease Plant Physiol. Vol. 107, 1995

imentally determining a necessary and sufficient PTS for plant peroxisomes.

mic phase (50 mL, 4 d posttransfer) were resusp3nded 1:l (v/v) in 2X transformation medium (2X growtb. medium without 2,4-D, plus 250 mM sorbitol and 250 mM rnannitol). Three pieces of Whatman No. 4 filter paper in the lid of a MATERIALS AND METHODS

Suspension-cultured tobacco cells (Nicotiana tabacum L. cv Bright Yellow 2 [Nagata et al., 199211, kindly provided by R. Cyr (Pennsylvania State University, University Park), were maintained on a orbital shaker at 25°C in the dark in 125-mL Erlenmeyer flasks containing Murashige-Skoog medium. Cells were subcultured once a week. Protoplasts of these cells were generated during a 2-h period at 30°C with gentle shaking in a 100-mm Petri dish containing 4 mL of cells and 16 mL of digestion medium composed of 1% (w/v) cellulase Y-C, 0.1% (w/v) pectolyase Y-23 (both from Seishin Pharmaceutical Co., Tokyo, Japan), and 350 mM mannitol (pH 5 f 0.3) according to the method of Kuss- Wymer and Cyr (1992).

Vectors and Site-Directed Mutagenesis

pRTL2-GUS was a gift from J. Carrington (Texas A&M University, College Station, TX) (Restrepo et al., 1990). pCAMVCN, purchased from Pharmacia, encoded CAT. The CAT DNA in this plasmid was modified by appending DNA encoding SKL to yield CAT-SKL (pCAMVC-SKL-N) via overlap extension PCR (Ho et al., 1989; Trelease et al., 1994). The first round, consisting of two separate PCRs (25 cycles each) with 2.5 units of Amplitaq (Perkin-Elmer Ce- tus) per 100 pL reaction and 1.5 mM MgCI, (optimized), involved four different oligonucleotide primers (1 FM each/reaction) and pCAMVCN (30 ng/reaction) as tem- plate. Sequences within the forward (39-mer) and reverse (36-mer) nonmutagenic primers were complementary to unique restriction sites (NcoI and CZaI) in pCAMVCN, whereas the reverse (5'-CTGCCTTATAATTTTGACGCC- 3') and forward (5'-GGCAGGGCGGGGCGTCAAAAT- TATAAGGCAGTTATTGG-3') mutagenic primers were complementary over the entire length (21 bases) of the reverse mutagenic primer. For the reaction with forward primer and the mutagenic 38-mer, the annealing temperature was 65"C, whereas it was 56°C for the other reaction. The two DNA fragments (200 and 400 bp) from the first two reactions were gel purified and collected on DEAE membranes, eluted at 68°C (1 h) into 10 mM Tris-HCI, 1 mM EDTA, 100 mM NaC1, pH 8.0, and then concentrated in a Centricon-30 (Amicon, Beverly, MA) microconcentrator. In the second-round PCR (25 cycles, 3.0 mM MgCl, [opti- mizedl, annealing temperature 56"C), 50 ng of the 200-bp and 100 ng of the 400-bp double-stranded fragments (tem- plates with 30-base overlap) were mixed with 1 PM each of the nonmutagenic primers. The final product, a 600-bp NcoI/CZaI DNA fragment, was gel purified, concentrated, and then ligated into pCAMVCN after both were double digested with NcoI and ClaI (4X enzyme concentration, overnight at 37°C).

Microprojectile Bombardment of TBY-2 Cells

Procedures were modified from Russell et al. (1992). Pelleted TBY-2 suspension cells collected in early logarith-

100- 15" Petri dish were moistened with 1.5 mL of transformation medium. Resuspended cells (2.5 mL) were spread dropwise from a 5-mL pipet in a thin layer onto the top filter paper. Cells were equilibrated at 25°C with the sugar alcohols for 1 h prior to bombardment. I3NA was precipitated onto tungsten particles by the calcium chlo- ride-spermidine method described in the Bio-Rad instruc- tion manual for the Biolistic Particle Deliver y System (1000/He). Cells placed at a 115-mm target dist,ince were bombarded at 1300 psi in 28-inch Hg vacuum with 0.25- inch gap and 1-cm flight distances.

lmmunofluorescent Microscopy

Bombarded cells were left in unwrapped dishes for 3 h (pRTL2-GUS) or 24 h (pCAMVCN or pCAMVC-SKL-N) at 25°C in a laminar flow hood with diffuse fluorescent light- ing. Cells in one plate routinely were bomba-ded with pRTL2-GUS on the same day as bombardments vvith other plasmids to determine the area on the filter paFers where cells were transiently transformed (determined by colorimetric GUS assay; Jefferson, 1987). Selected cells, gently scraped from the top filter paper with a spatuli (about 1 mL PCV), were fixed for 1 h at room temperature in about 9 mL of 4% formaldehyde (made from paraformaldehyde) in 1 X transformation buffer. Nontransformed cells (about 1 mL PCV) collected from culture flasks and washed in PBS (4.3 mM Na,HPO,, 1.4 mM KH,PO,, 2.7 mM KCl, and 137 mM NaC1, pH 7.4) were fixed for 1 h at room temperature in about 4 mL of 4% formaldehyde in PBS. One milliliter of gravity-settled TBY-2 protoplasts were fixed at 25°C in 3 mL of 4% formaldehyde, 350 mM mannitol, 50 m M sodium phosphate, pH 7.0.

The following procedures were followed for both transiently transformed and nontransformed cells. Fixed cells were washed by repeated centrifugations (about 600 rpm, 2-3 min, in a table-top clinical centrifuge), first in fixation buffer and then twice in water. Cells were resuspended and incubated in about 4 volumes of 0.1% (w/v) pectolyase Y-23 in water (pH 5 f 0.3) for 2 h at 30°C. After three washes in PBS, cells were permeabilized in about 5 volumes of 0.3% Triton X-100 (Sigma) in PBS for 15 min at room temperature. Pectolyase-treated cells may be stored for at least 1 week at 4°C prior to permeabilization. Perme- abilization of protoplasts was accomplished during the last 10 min of the fixation by addition of Triton X-100 to a final concentration of 0.1% (v/v).

Washed, permeabilized cells or protoplasts, separated into 250-pL PCV portions, either were brought to 1 mL with PBS plus anti-cottonseed catalase IgGs (prepared with protein A affinity columns; Kunce et al., 1988) (1:250 dilu- tion), or were resuspended in 250 pL of undiluted anti- CAT monoclonal antibodies (gift from S. Subramani, San Diego, CAI, or were incubated with both primary antibodies simultaneously (250 pL of anti-CAT, 2 pL of anti-cotton catalase) for 1 h at 25°C. After three PBS washes, cells (or

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In Vivo Peroxisome lmport System 1203

protoplasts) were brought to 1 mL in PBS plus TO pL of secondary antibodies, e.g. goat anti-rabbit FITC or TRITC (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and/or goat anti-mouse BODIPY (Molecular Probes, Inc., Eugene, OR) IgGs for 45 to 60 min at 25°C. Controls included applying primary and/or secondary antibodies to either nontransformed or mock-transformed (with linear calf thymus DNA) cells. Photographs were taken with Kodak TMZ 135 (~3200) black and white film using a Nikon Fluophot or Zeiss Axioskop microscope.

Assays and lmmunoblot Analysis

Catalase activity was measured according to the method of Ni et al. (1990). TBY-2 extracts were prepared with a French pressure cell (one pass, 800 psi, 3.5-mL pressure cell). For SDS-PAGE and subsequent electroblotting (Cor- pas et al., 19941, protein in the extracts had to be concentrated; this was accomplished by following the initial steps in catalase purification described by Ni et al. (1990) for purification of cottonseed catalase. Protein content was determined by the Bradford (1976) procedure with bovine y-globulin as the standard.

RESULTS AND DISCUSSION

Evaluation of Protoplasts as an lmport System

It was believed initially that the most suitable plant in vivo import system would be one that required preparation of protoplasts. Toward that end, protoplasts were prepared from leaves of cotton and tobacco explants and from severa1 suspension-cultured cell lines, namely cotton, carrot, alfalfa, and tobacco BY-2. Protoplast preparation and/or immunofluorescent staining of endogenous catalase proved to be unsuitable for most of these cell systems (details are not discussed). Figure 1 A, however, shows an example of the most desirable protoplast system that was studied. Indirect immunofluorescent staining of TBY-2 protoplasts with anti-cottonseed catalase as the primary anti- body yielded a clearly observable punctate pattern throughout the cytoplasm of essentially a11 protoplasts. Such images were similar to the immunofluorescent detection of catalase and other proteins in peroxisomes of different cultured mammalian (Gould and Subramani, 1991; Subramani, 1993) and yeast (Aitchison et al., 1992; Zhang et al., 1993; Erdman, 1994) cells. The results showed the fea- sibility of evaluating, by immunofluorescent microscopy, in vivo targeting and import of proteins into putative TBY-2 peroxisomes.

Evaluations of Nontransformed and Microprojectile- Bombarded TBY-2 Cells

To our knowledge, activity of catalase, or any other peroxisomal marker enzyme in TBY-2 cells, has not been reported. Assays for catalase activity in TBY-2 extracts

yielded a specific activity of 0.2 pkat mg-’ protein, which was about 12% of that measured, for comparison, in cotyledon extracts of 2-d-old cotton seedlings. Comparative western blot analyses of extracts from cotton cotyledons and TBY-2 cells revealed that the affinity-purified, anti- cotton catalase IgGs used for our immunofluorescent studies recognized a 57-kD polypeptide in cotton extracts (as reported by Kunce et al., 1988) and two polypeptides of approximately 57 and 59 kD in the TBY-2 cell extracts (data not shown). Catalase subunits of the same two molecular masses were reported for sunflower cotyledon catalase (Eising et al., 1990). Collectively, these results indicate that authentic catalase exists in TBY-2 cells and that it is immu- noreactive with anti-cottonseed catalase IgGs.

Although satisfactory immunofluorescent results were obtained with TBY-2 protoplasts (Fig. lA), problems were often experienced relative to reproducibility and handling of the fragile protoplasts. This prompted further experi- mentation, which led to the discovery that reliable, high- quality images of anti-catalase immunofluorescence could be obtained with TBY-2 cells that were not converted completely into protoplasts but were treated with pectolyase only. An example of catalase staining in such nontransformed cells (permeabilized with 0.3% Triton X-100) is shown in Figure 1B. The dependence of the punctate images on application of anti-catalase antibodies is shown in Figure 1C. Figure 1D shows that, if cells are not treated with pectolyase, but are permeabilized with Triton X-100 (0.3%), visualization of putative peroxisomes is apparent in most cells, but the intensity of the images is substantially decreased. Essentially the same results were obtained if cells were treated with pectolyase but not permeabilized with Triton X-100 (examples not shown). Immunofluores- cence within a11 cells was abolished if both treatments (pectolyase and Triton X-100) were omitted (not shown). Thus, at least partial cell wall digestion and detergent permeabilization of cell membranes were required to ob- tain optimal intensity of fluorescent, putative peroxisome images with any of the conjugated fluorochromes that we tested (i.e. FITC, TRITC, BODIPY).

Immunofluorescent imaging of putative peroxisomes with anti-catalase antibodies was the same in cells sub- jected to microprojectile bombardment and subsequent equilibration in isoosmotic tranformation medium for up to 48 h. Figure 1F shows distinct immunofluorescent, endogenous catalase-stained particles in the cytoplasmic strips bordering the central vacuole in bombarded cells equilibrated for 24 h prior to formaldehyde fixation. Vir- tually a11 cells exhibited this pattern. If fixed cells were treated with pectolyase, but were not permeabilized with Triton X-100, then the quality of the images was decreased (Fig. 1E). A weak punctate pattern was observed in some cells, but most appear as shown with a low-intensity fluo- rescence throughout the cytoplasm, similar to cells shown in Figure 1D. It seems that pectolyase treatment contributes to partial permeabilization of the plant cell membranes (the same results were obtained with nontransformed cells), but the mechanism(s) is not known by us or, to our knowledge, reported or discussed in the literature.




Figure 1. Immunofluorescent localization of catalase in nontransformed (A-D) and transiently transformed (E and F) TBY-2cells visualized by anti-cottonseed catalase IgCs (1:500 for A and 1:250 for B and D-F) bound to fluorochrome-conjugatedgoat anti-rabbit IgCs (1:100). A, TBY-2 protoplasts permeabilized with 0.1% Triton X-100 exhibit a punctate, cytosolicimmunofluorescence (FITC) pattern showing the distribution of putative peroxisomes. B and C, Cells treated with pectolyaseand 0.3% Triton X-100 exhibit putative peroxisomes stained with TRITC in B but not in C, where anti-catalase IgGs werenot applied. D, Cells immunostained as in B but not previously incubated in pectolyase; visualization of the punctate patternis greatly reduced. E and F, Cells transiently transformed with pCAMVC-SKL-N and processed for visualizing endogenouscatalase as for B, except cells in E were not permeabilized with Triton X-100. Bar = 10 /xm (same for all photographs).

Total incubation time in pectolyase is critical for optimalstaining of transformed and nontransformed cells. Non-transformed cells examined after 0.5-h intervals during a2-h period showed an increased percentage of the cellswith a complete punctate pattern (all cells were permeabi-lized with Triton X-100) (data not shown). After at least 2 h,virtually all of the cells in each microscopic field (X400)exhibited the optimal punctate pattern. The concentrationof Triton X-100 and the time of application also affects theimmunofluorescent images in both transformed and non-transformed cells (data not shown). Concentrations greaterthan 0.5% (v/v) for times longer than 30 min generallydecreased the quality of the images, most likely because of

solubilization/vesiculation of the plasma and intracellularmembranes.

Preference of Microprojectile Bombardment forIntroducing DNAs Encoding Modified Peroxisome Proteins

Microprojectile bombardment consistently produced agreater percentage of transiently transformed cells (esti-mated to be as high as 5%) when compared with results oftransformation with the 40% PEG method or by electropo-ration. This was especially evident when three consecutivebombardments were applied to the plates of cells that wererotated 120 degrees between each bombardment to mini- www.plantphysiol.orgon September 10, 2020 - Published by Downloaded from

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In Vivo Peroxisome Import System 1205

mize cell destruction. When cells were bombarded four orfive times consecutively, the percentage of surviving trans-formants noticeably decreased. Under our conditions, ex-pression of GUS was detectable by colorimetric staining ofcells within 2.5 h of the final bombardment with pRTL2-GUS. Expression within a short time is likely due to acombination of biolistically transforming the cells and us-ing the pRTL2-based vector possessing an mRNA transla-tion enhancer (Carrington and Freed, 1990). Expression ofCAT-SKL from pCAMVC-SKL-N was not detectable after 4h but consistently was immunostained 24 h after the finalbombardment. This differential in time of expression isadvantageous because one can determine the area on thefilter paper where the cells possess the introduced DNA onthe same day as the bombardment and then process thecells the next day to learn the results of import experi-ments. Even with selection of transiently transformed cellsby parallel GUS activity staining, only a low percentage(about 1-5%) of the cells express the protein of interest.This precludes immunocytochemical observations and/orbiochemical assays of isolated TBY-2 peroxisomes to con-firm the immunofluorescent localizations that are easilyobserved among the thousands of cells on a slide under a20- x 40-mm coverslip.

Co-Localization of Endogenous Catalase withCAT-SKL in Peroxisomes

The catalase-stained particles observed in the cellsshown in Figure 1 likely are peroxisomes, but confirmationis needed. A method commonly used is to co-localize anendogenous marker protein with an introduced protein tothe same organelle. This has been accomplished in culturedmammalian cells by double-label indirect immunofluores-cence of endogenous catalase with firefly luciferase (Kelleret al., 1987; Gould et al., 1987,1988), CAT-SKL (Gould et al.,1989), CAT-PMP20 fusion protein (Gould et al., 1990a,1990b), and cottonseed ICL (Trelease et al., 1994). It alsowas done with transgenic tobacco plant cells in whichendogenous glycolate oxidase was co-localized with intro-duced castor bean ICL (Marrison et al., 1993).

We chose to authenticate the putative TBY-2 peroxi-somes by co-localization of endogenous catalase with in-troduced CAT-SKL (Fig. 2). A previous study showed thatproteins with a C-terminal SKL occurred in castor beanglyoxysomes (Keller et al., 1991). In Figure 2A, only one ofseveral cells exhibits putative peroxisomes stained withanti-CAT-SKL antibodies in the nonvacuolar, cytoplasmicarea of the cell. These organelles are visible with the fluo-

Figure 2. Immunofluorescent localizations of CAT-SKL (A and C) and catalase (D) in pectolyase- and Triton X-100-treatedTBY-2 cells transiently transformed with pCAMVC-SKL-N. A and B, One of several cells in A reveals a punctate, putativeperoxisomal staining pattern (arrow) after applications of mouse anti-CAT IgGs (undilute) and goat anti-mouse BODIPY IgGs(1:100) and viewing with the fluorescein filter, whereas the same cells viewed with the rhodamine filter do not reveal anyimmunofluorescence (B). C and D, Co-localization of punctate CAT-SKL (C) with punctate endogenous catalase in the samecell (D). Cells in C were viewed with a fluorescein filter and the same cells in D were viewed with a rhodamine filter.Antibodies added to the cells were those described for A, plus rabbit anti-cottonseed catalase IgGs (1:250) and goatanti-rabbit-TRITC IgGs (1:100). Note: All cells in D reveal a punctate immunofluorescence due to catalase staining, whereasonly one cell shows a punctate pattern in C, which is identical with the same cell viewed in D. Bar = 10 /xm (same for allphotographs).




rescein filter after application of goat anti-mouse antibod-ies conjugated to BODIPY but, as expected, are not visiblewhen the same cells are viewed with the rhodamine filter(Fig. 2B). Figure 2, C and D, shows cells on a different slidestained with the mouse monoclonal antibodies and rabbitanti-cotton catalase IgGs. In Figure 2C, putative peroxi-somes stained with CAT-SKL antibodies are observed withthe fluorescein filter in the cytoplasm of one cell, and inFigure 2D the same organelles stained with catalase anti-bodies are observed with the rhodamine filter. A surfaceview of the cell's cytoplasm was photographed to illustratethe superimposable punctate patterns. Convincing evi-dence that the image in Figure 2D is due to endogenouscatalase is that all cells exhibit a punctate pattern, not justone transiently transformed cell as shown in Figure 2C.

A current limitation of the system, however, is that wehave not unequivocally demonstrated that the CAT-SKL istranslocated into the peroxisomes, i.e. the punctate immu-nofluorescent pattern could be due to antibodies binding toproteins on the cytosolic side of the peroxisome boundarymembranes. A method commonly used to show importinto mammalian peroxisomes is by differential treatmentwith detergents, namely digitonin to selectively permeabi-lize the plasma membrane with and without low concen-trations of Triton X-100 to permeabilize peroxisome mem-branes (Swinkels et al., 1991; Walton et al., 1992; Soto et al.,1993; Trelease et al., 1994). We have attempted similarexperiments with the TBY-2 cells with unpredictable re-sults, probably because of the difference in sterol content inplant and mammalian plasma membranes and to the ap-parent partial membrane permeabilization resulting frompectolyase treatment. Experiments are in progress to find amethod that will allow us to determine definitivelywhether introduced proteins are imported. Our currentbelief, based on our collective results and those in theliterature, is that CAT-SKL is translocated into the plantperoxisome matrix.

Evidence That SKL-COOH Is a Targeting Signal forPlant Peroxisomes

The co-localization of CAT-SKL with endogenous cata-lase shown in Figure 2 suggests that transiently expressedCAT-SKL is targeted to TBY-2 peroxisomes. Figure 3 pro-vides direct evidence for this conjecture. Figure 3A showsthat peroxisomes are stained with antibodies to CAT whenthe cells are transiently transformed with DNA encodingCAT-SKL (similar to results shown in Fig. 2, A and C).When CAT without the appended C-terminal SKL(—EWQGGA-COOH) is transiently expressed in cells (Fig.3B), a distinctly different pattern is observed. The fluores-cent signal occurs throughout the nonvacuolar cytoplasm,which is indicative of CAT expression without import intoperoxisomes. Thus, the C-terminal GGA on the CAT pro-tein, which is not a PTS1 type of signal, does not functionas a targeting signal to plant peroxisomes. The same twoimmunofluorescent patterns were observed in suspension-cultured carrot cells transiently transformed with eitherCAT or CAT-SKL (data not shown). If the primary anti-bodies are not applied, a fluorescent image is not observed(Fig. 3C).

Experimental evidence for a C-terminal SKL, or anyother tripeptide, being necessary or sufficient for targetingproteins in vivo or in vitro to plant peroxisomes had notbeen reported. Firefly luciferase, with a C-terminal SKLessential for import into mammalian cells (Gould et al.,1989; Swinkels et al., 1992), was imported into leaf peroxi-somes of transgenic tobacco plants, but mutagenesis exper-iments were not done to determine which portion of theenzyme was necessary for import. As mentioned above,antibodies to a peptide containing a C-terminal SKL recog-nized numerous castor bean glyoxysome proteins on west-ern blots and bound to protein in the matrix of castor beanglyoxysomes via immunocytochemistry (Keller et al.,1991), but the necessity of the C-terminal tripeptide was not

Figure 3. Immunofluorescent localization of CAT-SKL (A) and CAT (B) in TBY-2 cells transiently transformed withpCAMVC-SKL-N and pCAMVCN, respectively. Cells in both groups were treated and viewed as for Figure 2A. Onetransformed cell in A reveals a punctate pattern indicative of peroxisome localization, whereas one cell in B showsnonpunctate immunofluorescence throughout the nonvacuolar portion of the cytoplasm. C, Cells treated and viewed as forA, except that the mouse anti-CAT antibodies were not applied. Bar = 10 im (same for all photographs).



In Vivo Peroxisome lmport System 1207

demonstrated. In other studies, castor bean ICL-SKL- COOH, rapeseed ICL-SRM-COOH, and oilseed rape malate synthase SKL-COOH were imported into transgenic tobacco leaf or Avabidopsis leaf and root peroxisomes (Mar- rison et al., 1993; Olsen et al., 1993; Onyeocha et al., 1993), but only Olsen et al. (1993) and Volikita (1991) showed that specific C-terminal amino acids of oilseed rape ICL (CAT- AKSRM) and spinach glycolate oxidase (GUS-RAVARL), respectively, were sufficient for import.

The results reported here clearly show that TBY-2 cells simply treated with pectolyase and permeabilized with Triton X-100 can serve as a n effective and reliable in vivo import system to evaluate peroxisome-targeting signals on transiently expressed proteins. Our demonstration that the C-terminal SKL tripeptide is necessary and sufficient for in vivo targeting of at least CAT to TBY-2 suspension-cultured cell peroxisomes is in concert with studies showing that this tripeptide directs targeting to isolated liver peroxisomes (Miyazawa et al., 19891, to peroxisomes in cultured mammalian (Gould et al., 1989) and yeast (Distel et al., 1992) cells, and to glycosomes of trypanosomes (Fung and Clayton, 1991; Blattner et al., 1992; Sommer et al., 1992).

ACKNOWLEDCMENTS

We sincerely thank Drs. D. Capco and R. Roberson for the use of their fluorescent microscopes, Dr. C. Zeiher for her guidance with protoplast protocols, Dr. R. Cyr for his advice concerning handling and protoplasting TBY-2 cells, Tom Collela for his extensive tech- nical contributions, Jeff Bunkelmann for numerous supportive dialogues and help with electrophoresis, electoblotting, and chemiluminescence, and Dr. W. Xie for his insightful discussions during the course of this project.

Received November 16, 1994; accepted December 4, 1994. Copyright Clearance Center: 0032-0889/95/107/1201/08.

LITERATURE ClTED

Aitchison JD, Szilard RK, Nuttley WM, Rachubinski (1992) An- tibodies directed against a yeast carboxyl-terminal peroxisomal targeting signal specifically recognize peroxisomal proteins from various yeasts. Yeast 8 721-734

Beevers H (1979) Microbodies in higher plants. Annu Rev Plant Physiol80: 159-193

Blattner J, Swinkels B, Dorsam H, Prospero T, Subramani S, Clayton C (1992) Glycosome assembly in trypanosomes: varia- tions in the acceptable degeneracy of a COOH-terminal microbody targeting signal. J Cell Biolll9: 1129-1136

Borst P (1989) Peroxisome biogenesis revisited. Biochim Biophys Acta 1008 1-13

Bradford MM (1976) A rapid and sensitive method for the quan- tification of microgram quantities of proteins utilizing the prin- ciple of protein-dye binding. Ana1 Biochem 72 248-254

Carrington JC, Freed DD (1990) Cap-dependent enhancement of translation by a plant potyvirus 5’ nontranslated region. J Virol

Corpas FJ, Bunkelmann J, Trelease RN (1994) Identification and immunochemical characterization of a family of peroxisome membrane proteins (PMPs) in oilseed glyoxysomes. Eur J Cell Biol 65: 280-290

de Hoop MJ, Ab G (1992) Import of proteins into peroxisomes and other microbodies. J Biochem 286 657-669

Distel B, Gould SJ, Voom-Brouwer T, van der Berg M, Tabak HF, Subramani S (1992) The carboxyl-terminal tripeptide

6 4 1590-1597

serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast. New Biol 4: 157-165

Eising R, Trelease RN, Ni W (1990) Biogenesis of catalase in glyoxysomes and leaf-type peroxisomes of sunflower cotyledons. Arch Biochem Biophys 278 258-264

Erdman R (1994) The peroxisomal targeting signal of 3-oxoacyl- COA thiolase from Saccharomyces cerevisiae. Yeast 10 935-944

Fung K, Clayton C (1991) Recognition of a peroxisomal tripeptide entry signal by the glycosomes of Trypanosoma brucei. Mo1 Bio- chem Parasito1 45: 261-264

Gietl C, Faber KN, van der Klei IJ, Veenhuis M (1994) Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using heterologous host Hansenula polymorpha. Proc Natl Acad Sci USA 91: 3151-3155

Gould SJ, Keller GA, Hosken N, Wilkinson J, Subramani S (1989) A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 108: 1657-1664

Gould SJ, Keller GA, Schneider M, Howell SH, Garrard LJ, Goodman JM, Distel B, Tabak H, Subramani S (1990a) Perox- isomal protein import is conserved between yeast, plants, in- sects and mammals. EMBO J 9: 85-90

Gould SJ, Keller GA, Subramani S (1987) Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol 105: 2923-2931

Gould SJ, Keller GA, Subramani S (1988) Identification of peroxisomal targeting signals located at the carboxy tenninus of four peroxisomal proteins. J Cell Biol 107: 897-905

Gould SJ, Krisans S , Keller GA, Subramani S (1990b) Antibodies directed against peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins. J Cell Biol

Gould SJ, Subramani S (1991) Translocation of proteins into peroxisomes. In C Steer, J Hanover, eds, Intracellular Trafficking of Proteins. Cambridge University Press, Cambridge, UK, pp

Ho SN, Hunt HD, Morton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the poly- merase chain reaction. Gene 77: 51-59

Huang AHC, Trelease RN, Moore TS (1983) Plant Peroxisomes. American Society of Plant Physiologists Monograph Series. Ac- ademic Press, New York

Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mo1 Biol Rep 5: 87405

Keller GA, Gould S, DeLuca M, Subramani S (1987) Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc Natl Acad Sci USA 8 4 3264-3268

Keller GA, Krisans S, Gould SJ, Sommer JM, Wang CC, Schliebs W, Kunau W, Brody S, Subramani S (1991) Evolutionary con- servation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes. J Cell Biol 114

Kindl H (1982) The biosynthesis of microbodies (peroxisomes, glyoxysomes). Int Rev Cytol 80: 193-229

Kunce CM, Trelease RN, Turley RB (1988) Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase. Bio- chem J 251: 147-155

Kuss-Wymer CL, Cyr RJ (1992) Tobacco protoplasts differentiate into elongate cells without microtubule depolymerization. Pro- toplasma 168: 64-72

Lazarow PB, Fujiki F (1985) Biogenesis of peroxisomes. Annu Rev Cell Biol 1489-530

Marrison JL, Onyeocha I, Baker A, Leech RM (1993) Recognition of peroxisomes by immunofluorescence in transformed and un- transformed tobacco cells. Plant Physiol 103: 1055-1059

Miura S, Kasuya-Arai AI, Mori H, Miyazawa S, Osumi T, Hashi- moto T, Fujiki Y (1992) Carboxyl-terminal consensus Ser-Lys- Leu-related tripeptide of peroxisomal proteins function in vitro as a minimal peroxisome-targeting signal. J Biol Chem 267: 14405-14411

Miyazawa S , Osumi T, Hashimoto T, Ohno K, Miura S , Fujiki Y (1989) Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. Mo1 Cell Biol 9: 83-91

110: 27-34

697-730

893-904




Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the "HeLa" cell in the cell biology of higher plants. Int Rev Cytol

Ni W, Trelease RN, Eising R (1990) Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirustum) catalase. Biochem J 269 233-238

Olsen LJ, Ettinger WF, Damsz B, Matsudaira K, Webb MA, Harada JJ (1993) Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant. Plant Cell5 941-952

Onyeocha I, Behari R, Hill D, Baker A (1993) Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes. Plant Mo1 Biol 22 385-396

Osumi T, Tsukamoto T, Hata S, Yokota S, Fujiki Y, Miyazawa S, Hashimoto T (1991) Amino-terminal presequence of the precur- sor of peroxisomal 3-ketoacyl-COA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem Biophys Res Com- inun 181: 947-954

Restrepo MA, Freed DD, Carrington JC (1990) Nuclear transport of plant potyviral proteins. Plant Cell2 987-998

Russell JA, Mihir KR, Sanford JC (1992) Major improvements in biolistic transformation of suspension-cultured tobacco cells. In Vitro 28: 97-105

Sommer JM, Cheng Q-L, Keller G-A, Wang CC (1992) In vivo import of firefly luciferase into the glycosomes of Typunosomu brucei and the mutational analysis of the C-terminal targeting signal. Mo1 Biol Cell 3: 749-759

Soto U, Pepperkok R, Ansorge W, Just WW (1993) Import of firefly luciferase into mammalian peroxisomes in vivo requires

132: 1-30

nucleoside triphosphates. Exp Cell Res 205: 66-75 Subramani S (1993) Protein import into peroxisomes and biogen-

esis of the organelle. Annu Rev Cell Biol 9 445-478 Swinkels SW, Gould SJ, Bodnar AG, Rachubinski KA, Subra-

mani S (1991) A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-COA thio1a:;e. EMBO J

Swinkels SW, Gould SJ, Subramani S (1992) Targetiiig efficien- cies of various permutations of the consensus C-terminal tripeptide peroxisomal targeting signal. FEBS Lett 305: 133-136

Trelease RN, Choe SM, Jacobs BL (1994) Conserva:ive amino acids substitutions of the C-terminal tripeptide, ARM, on cottonseed (Gossypium hirustum L.) isocitrate lyase permi t import in vivo into mammalian cell peroxisomes. Eur J Cell Biol 65:

van den Bosch H, Schutgens RBH, Wanders RJA, Tage r JM (1992) Biochemistry of peroxisomes. Annu Rev Biochem 61: 157-197

Volikita M (1991) The carboxy-terminal end of glycolate oxidase directs a foreign protein into tobacco leaf peroxisome;. Plant J 1:

Walton PA, Gould SJ, Feramisco JR, Subramani S (1992) Trans- port of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal. Mo1 Cc11 Biol 1 2

Zhang JW, Han Y, Lazarow PB (1993) Nove1 peroxisorne cluster- ing mutants and peroxisome biogenesis mutants of Siccharomy- ces cerevisiue. J Cell Biol 123: 1133-1147

10: 3255-3262

269-279

361-366

531-541



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