Richard N. Trelease

Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes.

Isocitrate lyase (IL) is an essential enzyme in the glyoxylate cycle, which is a pathway involved in the mobilization of stored lipids during postgerminative growth of oil-rich seedlings. We determined experimentally the necessary and sufficient peroxisome targeting signals (PTSs) for cottonseed, oilseed rape, and castor bean ILs in a well-characterized in vivo import system, namely, suspension-cultured tobacco (Bright Yellow) BY-2 cells. Results were obtained by comparing immunofluorescence localizations of wild-type and C-terminal-truncated proteins transiently expressed from cDNAs introduced by microprojectile bombardment. The tripeptides ARM-COOH (on cottonseed and castor bean ILs) and SRM-COOH (on oilseed rape IL) were necessary for targeting and actual import of these ILs into glyoxysomes, and ARM-COOH was sufficient for redirecting chloramphenicol acetyltransferase (CAT) from the cytosol into the glyoxysomes. Surprisingly, IL and CAT subunits without these tripeptides were still acquired by glyoxysomes, but only when wild-type IL or CAT-SKL subunits, respectively, were simultaneously expressed in the cells. These results reveal that targeting signal-depleted subunits are being piggybacked as multimers to glyoxysomes by association with subunits possessing a PTS1. Targeted multimers are then translocated through membrane pores or channels to the matrix as oligomers or as subunits before reoligomerization in the matrix.

Peroxisomal Membrane Ascorbate Peroxidase Is Sorted to a Membranous Network That Resembles a Subdomain of the Endoplasmic Reticulum

The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD+ and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3′;-dihexyloxacarbocyanine iodide–stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted posttranslationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase–peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain.

Overexpression and mislocalization of a tail-anchored GFP redefines the identity of peroxisomal ER.

Peroxisomal ascorbate peroxidase (APX) sorts indirectly via a subdomain of the ER (peroxisomal ER) to the boundary membrane of peroxisomes in tobacco Bright Yellow 2 cells. This novel subdomain characteristically appears as fluorescent reticular/circular compartments distributed variously in the cytoplasm. Further characterizations are presented herein. A peptide possessing the membrane targeting information for peroxisomal APX was fused to GFP (GFP‐APX). Transiently expressed GFP‐APX sorted to peroxisomes and to reticular/circular compartments; in both cases, the GFP moiety faced the cytosol. Of particular interest, both homotypic and heterotypic aggregates of peroxisomes, mitochondria, and/or plastids were formed. The latter two organelles comprised the circular portion of the reticular/circular compartments, apparently as a consequence of oligomerization (zippering) of the GFP moieties after insertion into the outer membranes of the affected organelles. These results, coupled with the accumulation of endogenous peroxisomal APX in cytoplasmic, noncircular compartment(s) following treatment with brefeldin A, indicate that authentic peroxisomal ER is composed only of a reticular compartment(s). Equally important, the data show that overexpressed, membrane‐targeted GFP fusion proteins have a propensity to form organelle aggregates that may lead to misinterpretations of sorting pathways of trafficked proteins.

Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A Cooperate in Cell Cycle–Associated Replication of Peroxisomes

Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle–associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in ∼40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells.

The Sorting Signals for Peroxisomal Membrane-bound Ascorbate Peroxidase Are within Its C-terminal Tail

Peroxisomal ascorbate peroxidase (APX) is a carboxyl tail-anchored, type II (Ncytosol-Cmatrix) integral membrane protein that functions in the regeneration of NAD+ in glyoxysomes of germinated oilseeds and protection of peroxisomes in other organisms from toxic H2O2. Recently we showed that cottonseed peroxisomal APX was sorted post-translationally from the cytosol to peroxisomes via a novel reticular/circular membranous network that was interpreted to be a subdomain of the endoplasmic reticulum (ER), named peroxisomal ER (pER). Here we report on the molecular signals responsible for sorting peroxisomal APX. Deletions or site-specific substitutions of certain amino acid residues within the hydrophilic C-terminal-most eight-amino acid residues (includes a positively charged domain found in most peroxisomal integral membrane-destined proteins) abolished sorting of peroxisomal APX to peroxisomes via pER. However, the C-terminal tail was not sufficient for sorting chloramphenicol acetyltransferase to peroxisomes via pER, whereas the peptide plus most of the immediately adjacent 21-amino acid transmembrane domain (TMD) of peroxisomal APX was sufficient for sorting. Replacement of the peroxisomal APX TMD with an artificial TMD (devoid of putative sorting sequences) plus the peroxisomal APX C-terminal tail also sorted chloramphenicol acetyltransferase to peroxisomes via pER, indicating that the peroxisomal APX TMD does not possess essential sorting information. Instead, the TMD appears to confer the proper context required for the conserved positively charged domain to function within peroxisomal APX as an overlapping pER sorting signal and a membrane peroxisome targeting signal type 2.

Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.

Diverse amino acid residues function within the type 1 peroxisomal targeting signal : Implications for the role of accessory residues upstream of the type 1 peroxisomal targeting signal

The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting. The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting. The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting. The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting.

Five Arabidopsis peroxin 11 homologs individually promote peroxisome elongation, duplication or aggregation

Pex11 homologs and dynamin-related proteins uniquely regulate peroxisome division (cell-cycle-dependent duplication) and proliferation (cell-cycle-independent multiplication). Arabidopsis plants possess five Pex11 homologs designated in this study as AtPex11a, -b, -c, -d and -e. Transcripts for four isoforms were found in Arabidopsis plant parts and in cells in suspension culture; by contrast, AtPex11a transcripts were found only in developing siliques. Within 2.5 hours after biolistic bombardments, myc-tagged or GFP-tagged AtPex11 a, -b, -c, -d and -e individually sorted from the cytosol directly to peroxisomes; none trafficked indirectly through the endoplasmic reticulum. Both termini of myc-tagged AtPex11 b, -c, -d and -e faced the cytosol, whereas the N- and C-termini of myc-AtPex11a faced the cytosol and matrix, respectively. In AtPex11a- or AtPex11e-transformed cells, peroxisomes doubled in number. Those peroxisomes bearing myc-AtPex11a, but not myc-AtPex11e, elongated prior to duplication. In cells transformed with AtPex11c or AtPex11d, peroxisomes elongated without subsequent fission. In AtPex11b-transformed cells, peroxisomes were aggregated and rounded. A C-terminal dilysine motif, present in AtPex11c, -d and -e, was not necessary for AtPex11d-induced peroxisome elongation. However, deletion of the motif from myc-AtPex11e led to peroxisome elongation and fission, indicating that the motif in this isoform promotes fission without elongation. In summary, all five overexpressed AtPex11 isoforms sort directly to peroxisomal membranes where they individually promote duplication (AtPex11a, -e), aggregation (AtPex11b), or elongation without fission (AtPex11c, -d).

Peroxisomal Ascorbate Peroxidase Resides within a Subdomain of Rough Endoplasmic Reticulum in Wild-Type Arabidopsis Cells

Previously we reported (R.T. Mullen, C.S. Lisenbee, J.A. Miernyk, R.N. Trelease [1999] Plant Cell 11: 2167–2185) that overexpressed ascorbate peroxidase (APX), a peroxisomal membrane protein, sorted indirectly to Bright Yellow-2 cell peroxisomes via a subdomain of the endoplasmic reticulum (ER; peroxisomal endoplasmic reticulum [pER]). More recently, a pER-like compartment also was identified in pumpkin (Cucurbita pepo) and transformed Arabidopsis cells (K. Nito, K. Yamaguchi, M. Kondo, M. Hayashi, M. Nishimura [2001] Plant Cell Physiol 42: 20–27). Here, we characterize more extensively the localization of endogenous Arabidopsis peroxisomal APX (AtAPX) in cultured wild-type Arabidopsis cells (Arabidopsis var. Landsberg erecta). AtAPX was detected in peroxisomes, but not in an ER subcompartment, using immunofluorescence microscopy. However, AtAPX was detected readily with immunoblots in both peroxisomal and ER fractions recovered from sucrose (Suc) density gradients. Most AtAPX in microsomes (200,000g, 1 h pellet) applied to gradients exhibited a Mg2+-induced shift from a distribution throughout gradients (approximately 18%–40% [w/w] Suc) to ≥42% (w/w) Suc regions of gradients, including pellets, indicative of localization in rough ER vesicles. Immunogold electron microscopy of the latter fractions verified these findings. Further analyses of peroxisomal and rough ER vesicle fractions revealed that AtAPX in both fractions was similarly associated with and located mostly on the cytosolic face of the membranes. Thus, at the steady state, endogenous peroxisomal AtAPX resides at different levels in rough ER and peroxisomes. Collectively, these findings show that rather than being a transiently induced sorting compartment formed in response to overexpressed peroxisomal APX, portions of rough ER (pER) in wild-type cells serve as a constitutive sorting compartment likely involved in posttranslational routing of constitutively synthesized peroxisomal APX.

Development and Application of an in Vivo Plant Peroxisome Import System

The 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. Several were found suitable, but based on a combination of criteria, suspension-cultured tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells (TBY-2) were chosen. 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 G as the probe. Indirect immunofluorescence microscopy with these immunoglobulins G 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% (w/v) pectolyase for 2 h was sufficient. Microprojectile bombardment proved superior for transient transformation of the TBY-2 cells with plasmids encoding [beta]-glucuronidase, or chloramphenicol acetyltransferase (CAT), or CAT with an added C-terminal tripeptide (CAT-SKL). C-terminal SKL is 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.