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 approximately 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.

Arabidopsis peroxin 16 trafficks through the ER and an intermediate compartment to pre-existing peroxisomes via overlapping molecular targeting signals.

Previously it has been shown that the endogenous Arabidopsis peroxin, AtPEX16, coexisted at steady state in membranes of the endoplasmic reticulum (ER) and peroxisomes. Herein, an ER-to-peroxisome trafficking pathway and the requisite molecular targeting signals for mycAtPEX16 transiently expressed in Arabidopsis and tobacco BY-2 suspension cells are described. Immunofluorescent mycAtPEX16 was observed initially in the cytosol (<2 h) and subsequently (2-4 h) in perinuclear/reticular ER and non-Golgi/non-peroxisome structures termed the ER-peroxisome intermediate compartment. After 4 h, all catalase- and ascorbate peroxidase-containing peroxisomes also possessed mycAtPEX16, indicative of mycAtPEX16 sorting to pre-existing peroxisomes. Incubations of bombarded cells at 15 degrees C, or in brefeldin A at 25 degrees C, resulted in accumulations of mycAtPEX16 within the ER. Following re-equilibration of cold-treated cells at 25 degrees C, or removal of brefeldin A, mycAtPEX16 was observed mainly in the ER-peroxisome intermediate compartment, and later within all of the peroxisomes in both species. Two internal membrane helices and the intervening sequence including the amino acid residues -VRS- were found necessary and sufficient for targeting AtPEX16 first to the ER and then to peroxisomes. Individual targeting signals for these organelles were indistinguishable, indicative of overlapping signal(s). In summary, the trafficking study of AtPEX16 revealed a dynamic link between the ER and pre-existing peroxisomes, which provided novel data in support of an upgraded semi-autonomous peroxisome model portraying participation of the ER in the sorting of certain peroxisome membrane proteins, such as AtPEX16, through an intermediate compartment to pre-existing plant peroxisomes.

The ER-peroxisome connection in plants: development of the "ER semi-autonomous peroxisome maturation and replication" model for plant peroxisome biogenesis.

The perceived role of the ER in the biogenesis of plant peroxisomes has evolved significantly from the original "ER vesiculation" model, which portrayed co-translational import of proteins into peroxisomes originating from the ER, to the "ER semi-autonomous peroxisome" model wherein membrane lipids and post-translationally acquired peroxisomal membrane proteins (PMPs) were derived from the ER. Results from more recent studies of various plant PMPs including ascorbate peroxidase, PEX10 and PEX16, as well as a viral replication protein, have since led to the formulation of a more elaborate "ER semi-autonomous peroxisome maturation and replication" model. Herein we review these results in the context of this newly proposed model and its predecessor models. We discuss also key distinct features of the new model pertaining to its central premise that the ER defines the semi-autonomous maturation (maintenance/assembly/differentiation) and duplication (division) features of specialized classes of pre-existing plant peroxisomes. This model also includes a novel peroxisome-to-ER retrograde sorting pathway that may serve as a constitutive protein retrieval/regulatory system. In addition, new plant peroxisomes are envisaged to arise primarily by duplication of the pre-existing peroxisomes that receive essential membrane components from the ER.

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).

Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase.

The H2O2 byproduct of fatty acid catabolism in plant peroxisomes is removed in part by a membrane-associated antioxidant system that involves both an ascorbate peroxidase and a monodehydroascorbate reductase (MDAR). Despite descriptions of 32-kDa MDAR polypeptides in pea and castor peroxisomal membranes and cDNA sequences for several 'cytosolic' MDARs, the genetic and protein factors responsible for peroxisomal MDAR function have yet to be elucidated. Of the six MDAR polypeptides in the Arabidopsis proteome, named AtMDAR1 to AtMDAR6 in this study, 47-kDa AtMDAR1 and 54-kDa AtMDAR4 possess amino acid sequences that resemble matrix (PTS1) and membrane peroxisomal targeting signals, respectively. Epitope-tagged versions of these two MDARs and a pea 47-kDa MDAR (PsMDAR) sorted in vivo directly from the cytosol to peroxisomes in Arabidopsis and BY-2 suspension cells, whereas AtMDAR2 and AtMDAR3 accumulated in the cytosol. The PTS1-dependent sorting of AtMDAR1 and PsMDAR to peroxisomes was incomplete (inefficient?), but was improved for PsMDAR after changing its PTS1 sequence from -SKI to the canonical tripeptide -SKL. A C-terminal transmembrane domain and basic cluster of AtMDAR4 were necessary and sufficient for targeting directly to peroxisomes. MDAR activity in isolated Arabidopsis peroxisomes was distributed among both water-soluble matrix and KCl-insoluble membrane subfractions that contained respectively 47- and 54-kDa MDAR polypeptides. Notably, a 32-kDa MDAR was not identified. Combined with membrane association and topological orientation findings, these results indicate that ascorbate recycling in Arabidopsis (and probably other plant) peroxisomes is coordinated through functionally redundant MDARs that reside in the membrane and the matrix of the organelle.

Arabidopsis peroxin 16 coexists at steady state in peroxisomes and endoplasmic reticulum.

Homologs of peroxin 16 genes (PEX16) have been identified only in Yarrowia lipolytica, humans (Homo sapiens), and Arabidopsis (Arabidopsis thaliana). The Arabidopsis gene (AtPEX16), previously reported as the SSE1 gene, codes for a predicted 42-kD membrane peroxin protein (AtPex16p). Lin et al. (Y. Lin, J.E. Cluette-Brown, H.M. Goodman [2004] Plant Physiol 135: 814-827) reported that SSE1/AtPEX16 was essential for endoplasmic reticulum (ER)-dependent oil and protein body biogenesis in peroxisome-deficient maturing seeds and likely also was involved in peroxisomal biogenesis based on localization of stably expressed green fluorescent protein::AtPex16p in peroxisomes of Arabidopsis plants. In this study with Arabidopsis suspension-cultured cells, combined in vivo and in vitro experiments revealed a novel dual organelle localization and corresponding membrane association/topology of endogenous AtPex16p. Immunofluorescence microscopy with antigen affinity-purified IgGs showed an unambiguous, steady-state coexistence of AtPex16p in suspension cell peroxisomes and ER. AtPex16p also was observed in peroxisomes and ER of root and leaf cells. Cell fractionation experiments surprisingly revealed two immunorelated polypeptides, 42 kD (expected) and 52 kD (unexpected), in homogenates and microsome membrane pellets derived from roots, inflorescence, and suspension cells. Suc-gradient purifications confirmed the presence of both 42-kD and 52-kD polypeptides in isolated peroxisomes (isopycnic separation) and in rough ER vesicles (Mg2+ shifted). They were found peripherally associated with peroxisome and ER membranes but not as covalently bound subunits of AtPex16p. Both were mostly on the matrix side of peroxisomal membranes and unexpectedly mostly on the cytosolic side of ER membranes. In summary, AtPex16p is the only authentic plant peroxin homolog known to coexist at steady state within peroxisomes and ER; these data provide new insights in support of its ER-related, multifunctional roles in organelle biogenesis.

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-microm 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.

Sorting pathway and molecular targeting signals for the Arabidopsis peroxin 3.

Peroxin 3 (Pex3p) has been identified and characterized as a peroxisomal membrane protein in yeasts and mammals. We identified two putative homologs in Arabidopsis (AtPex3p, forms 1 and 2), both with an identical cluster of positively charged amino acid residues (RKHRRK) immediately preceding one of the two predicted transmembrane domains (TMD1). In transiently transformed Arabidopsis and tobacco BY-2 suspension-cultured cells, epitope-tagged AtPex3p (form 2) sorted post-translationally from the cytosol directly to peroxisomes, the first sorting pathway described for any peroxin in plants. TMD1 and RKHRRK were necessary for targeting form 2 to peroxisomes and sufficient for directing chloramphenicol acetyltransferase to peroxisomes in both cell types. The N and C termini of AtPex3p (form 2) extend into the peroxisomal matrix, different from mammal and yeast Pex3 proteins. Thus, two authentic peroxisomal membrane-bound Pex3p homologs possessing a membrane peroxisomal targeting signal, the first one defined for a plant peroxin and for any Pex3p homolog, exist in plant cells.

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 > or =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.

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.

Copper-zinc superoxide dismutase is a constituent enzyme of the matrix of peroxisomes in the cotyledons of oilseed plants.

The number and type of isoforms of superoxide dismutase (SOD) and their activities were compared in mitochondria and peroxisomes isolated from cotyledons of three different oilseed seedlings. Mitochondrial and peroxisomal isoforms of SOD could be distinguished in nondenaturing polyacrylamide gels by their differential sensitivities to KCN and/or H2 O2 . The type of SOD was not the same for each organelle in each of the three oilseed species. For example, a single Mn-SOD was found in cotton and cucumber mitochondria, whereas four CuZn-SODs were present in mitochondria from sunflower. At least one CuZn-SOD isoform was found in the peroxisomes of all three species. Cucumber peroxisomes contained both a CuZn-SOD and a Mn-SOD, cotton peroxisomes contained a single CuZn-SOD, whilst four separate CuZn-SODs, but no Mn-SOD were found in sunflower peroxisomes. Using antibodies against CuZn-SOD from watermelon peroxisomes or from chloroplasts of Equisetum, a single polypeptide of c. 16·5 kDa was detected on immunoblots of peroxisomal fractions from the three species. Post-embedment, electron-microscopic double immunogold-labelling showed that CuZn-SOD, with malate synthase used as marker enzyme of peroxisomes, was localized in the matrix of these organelles of all three species. These results suggest that CuZn-SOD is a characteristic matrix enzyme of peroxisomes in oilseed cotyledons.