Efficient prenylation by a plant geranylgeranyltransferase-I requires a functional CaaL box motif and a proximal polybasic domain.

Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common alpha-subunit with farnesyltransferase (FTase) and has a distinct beta-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I beta-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase alpha-subunit (FTA) as bait. Sequence and structure analysis revealed that the core active site of GGT-I and FTase are very similar. AtGGT-IA/FTA and AtGGT-IB were co-expressed in baculovirus-infected insect cells to obtain recombinant protein that was used for biochemical and molecular analysis. The recombinant AtGGT-I prenylated efficiently CaaL box fusion proteins in which the a(2) position was occupied by an aliphatic residue, whereas charged or polar residues at the same position greatly reduced the efficiency of prenylation. A polybasic domain proximal to the CaaL box motif induced a 5-fold increase in the maximal reaction rate, and increased the affinity of the enzyme to the protein substrate by an order of magnitude. GGT-I retained high activity in a temperature range between 24 degrees C and 42 degrees C, and showed increased activity rate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-polymerase chain reaction, protein immuno-blots, and transient expression assays of green fluorescent protein fusion proteins show that GGT-IB is ubiquitously expressed in a number of tissues, and that expression levels and protein activity were not changed in mutant plants lacking FTase beta-subunit.

Prenylation of the floral transcription factor APETALA1 modulates its function.

The Arabidopsis MADS box transcription factor APETALA1 (AP1) was identified as a substrate for farnesyltransferase and shown to be farnesylated efficiently both in vitro and in vivo. AP1 regulates the transition from inflorescence shoot to floral meristems and the development of sepals and petals. AP1 fused to green fluorescent protein (GFP) retained transcription factor activity and directed the expected terminal flower phenotype when ectopically expressed in transgenic Arabidopsis. However, ap1mS, a farnesyl cysteine-acceptor mutant of AP1, as well as the GFP-ap1mS fusion protein failed to direct the development of compound terminal flowers but instead induced novel phenotypes when ectopically expressed in Arabidopsis. Similarly, compound terminal flowers did not develop in era1-2 transformants that ectopically expressed AP1. Together, the results demonstrate that AP1 is a target of farnesyltransferase and suggest that farnesylation alters the function and perhaps specificity of the transcription factor.

Functional requirement of plant farnesyltransferase during development in Arabidopsis.

Arabidopsis era1 was identified as an abscisic acid-hypersensitive mutant caused by disruptions or deletions of the gene for the beta subunit (AtFTB) of farnesyltransferase (FTase). The heterodimeric enzyme catalyzes the covalent attachment of the 15-carbon farnesyl diphosphate to the C terminus of regulatory proteins and is essential for growth in yeast. The first disruption of FTB in a multicellular context revealed several developmental and growth regulatory processes that require the function of FTase. The lack of FTase activity in the Arabidopsis era1-2 FTB deletion mutant resulted in enlarged meristems and organs, supernumerary organs in floral whorls, arrested development of axillary meristems, late flowering, and homeotic transformations of flowers. Complementation of era1-2 with LeFTB, the tomato gene for the beta subunit of FTase, restored a normal phenotype and confirmed that the lesion is in AtFTB alone. The effect of this lesion on control of meristem size and on developmental processes suggests the involvement of regulatory proteins that require farnesylation for their function. At least three distinct processes that require the function of FTase were identified: regulation of cellular differentiation in the meristems, meristem maintenance, and regulation of flower development. Together, these results provide a basis for future studies on the involvement of FTase in specific developmental processes and for structure-function analysis of FTase in vivo.

Carboxyl-methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein.

Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post-prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP-labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl-methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl-cysteine methyltransferase (PCM) activity that carboxyl-methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.

Lipid modifications of proteins - slipping in and out of membranes.

Protein lipid modification, once thought to act as a stable membrane anchor for soluble proteins, is now attracting more widespread attention for its emerging role in diverse signaling pathways and regulatory mechanisms. Most multicellular organisms have recruited specific types of lipids and a suite of unique enzymes to catalyze the modification of a select number of proteins, many of which are evolutionarily conserved in plants, animals and fungi. Each of the three known types of lipid modification - palmitoylation, myristylation and prenylation - allows cells to target proteins to the plasma membrane, as well as to other subcellular compartments. Among the lipid modifications, protein prenylation might also function as a relay between cytoplasmic isoprene biosynthesis and regulatory pathways that control cell cycle and growth. Molecular and genetic studies of an Arabidopsis mutant that lacks farnesyl transferase suggest that the enzyme has a role in abscisic acid signaling during seed germination and in the stomata. It is becoming clear that lipid modifications are not just fat for the protein, but part of a highly conserved intricate network that plays a role in coordinating complex cellular functions.

The prenylation status of a novel plant calmodulin directs plasma membrane or nuclear localization of the protein.

Post-translational attachment of isoprenyl groups to conserved cysteine residues at the C-terminus of a number of regulatory proteins is important for their function and subcellular localization. We have identified a novel calmodulin, CaM53, with an extended C-terminal basic domain and a CTIL CaaX-box motif which are required for efficient prenylation of the protein in vitro and in vivo. Ectopic expression of wild-type CaM53 or a non-prenylated mutant protein in plants causes distinct morphological changes. Prenylated CaM53 associates with the plasma membrane, but the non-prenylated mutant protein localizes to the nucleus, indicating a dual role for the C-terminal domain. The subcellular localization of CaM53 can be altered by a block in isoprenoid biosynthesis or sugar depletion, suggesting that CaM53 activates different targets in response to metabolic changes. Thus, prenylation of CaM53 appears to be a novel mechanism by which plant cells can coordinate Ca2+ signaling with changes in metabolic activities.

Plant farnesyltransferase can restore yeast Ras signaling and mating.

Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes.

Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells.

Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport. To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase). We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells. The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence. LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase. LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato. LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit. The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening.

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif.

Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.

Accumulation of a light-harvesting chlorophyll a/b protein in the chloroplast grana lamellae. The lateral migration of the membrane protein precursor is independent of its processing.

The events that follow the import of pLHCPIIb, the apoprotein precursor of the major light-harvesting complex of photosystem II, were studied in intact pea chloroplasts. The distribution of the events of insertion into the membrane, and processing, to yield the mature form (LHCP) between stromal and granal lamellae regions of the thylakoids were followed. pLHCP was preferentially inserted into stromal lamellae (SL) from which it migrated to granal lamellae (GL). Migration occurred before or after processing, suggesting that migration and processing are independent of each other. When migration was slowed down, LHCP accumulated in SL. Prolonged inhibition of migration induced degradation of LHCP that had accumulated in SL, whereas inhibition of processing did not affect the migration of pLHCP into GL. A small difference in electrophoretic mobility was noted between LHCP in SL and in GL. The predominant mature form in SL migrated more slowly than LHCP from GL. When thylakoids were subjected to trypsin, all of the LHCP embedded in SL underwent cleavage, whereas up to 60% of the radioactive LHCP in GL was resistant to the enzyme. The possible implications of the differences in size and in the sensitivity to trypsin of LHCP are discussed.

Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane.

Molecular chaperones, including those belonging to the 70-kDa family of heat shock proteins (HSP70), assist both the translocation of proteins across membranes and their assembly into oligomeric complexes. We purified a chloroplast HSP70 (ct-HSP70) and demonstrated that it plays a major role in the insertion of the precursor of the major light-harvesting complex of photosystem II (pLHCP; an integral membrane protein) into the thylakoids (the inner membranes of the chloroplast). Addition of the purified ct-HSP70 is necessary for efficient insertion of pLHCP into isolated thylakoid membranes. This activity of the purified ct-HSP70 is similar to that previously reported for the total stromal extract. When the chloroplast stromal extract is depleted of HSP70, a correlative reduction in the insertion activity of pLHCP is observed. The interaction between the ct-HSP70 and pLHCP involves physical association. The purified HSP70 acts directly on the membrane protein, presumably prevents its refolding, and thereby helps to maintain its competence for insertion into membranes.

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.

On some of the in organello processes involved in the biogenesis of chlorophyll-protein complexes.

The biogenesis and assembly of chlorophyll-protein complexes consist of many steps. These are initiated with the transcription and translation of the different polypeptide components constituting the complexes. For the nuclear-encoded subunits the synthesis takes place in the cytoplasm, and they are synthesized as precursors, which are later imported into the chloroplast. Within the organelle, the precursors are inserted into the thylakoid membranes, as well as being processed to their mature forms. The different nuclear- and chloroplast-encoded subunits assemble together, and bind the pigments and other cofactors to form the active pigmented-complex. In the present article, we discuss only the in organello processes of the biogenesis. We describe the pathways taken by two nuclear-encoded thylakoid proteins, the precursor of the main light-harvesting chlorophyll-protein of photosystem II (pLHCP) and the precursor of photosystem I subunit II (pre subunit II). These polypeptide subunits, that are located in two different photosynthetic complexes, differ from each other. While pLHCP is an integral membrane protein, which binds pigments, photosystem I-subunit II is a peripheral membrane protein, located on the stromal side of the thylakoids, and is not predicted to span it. The differences and the common features of the in organello biogenesis pathways of these two proteins are discussed.

The apoprotein precursor of the major light-harvesting complex of photosystem II (LHCIIb) is inserted primarily into stromal lamellae and subsequently migrates to the grana.

The formation of the lateral distribution of the major antenna complex of photosystem II (LHCIIb) between the granal and stromal lamellae was studied. Specifically, the localization of the insertion and the assembly of the precursor of the apoprotein of LHCIIb (pLHCP) were studied with isolated thylakoids. After insertion of pLHCP into isolated thylakoids, fractionation of the latter into granal and stromal lamellar was performed. At 25 degrees C most of the precursor was located in the granal lamellae, although both highly purified granal and stromal lamellar fractions demonstrated a similar capability to insert pLHCP. When the insertion reaction to the thylakoids was performed at 10 degrees C, followed by their separation into stromal and granal lamellae, the labelled pLHCP was localized in the stromal ones. To examine whether pLHCP inserts into both granal and stromal lamellae, or preferentially into stromal lamellae and subsequently migrating to granal lamellae, a chase experiment was performed. Insertion of pLHCP at 10 degrees C was followed by chase of the radioactive precursor with excess of non-radioactive pLHCP at 25 degrees C. From the results presented it is evident that the level of pLHCP in stromal lamellae was gradually reduced, while it gradually accumulated in the granal lamellae. Furthermore, the pLHCP in the stromal lamellae was found to be in a 'free' form, while after migrating to the granal lamellae it assembled into the pigmented LHCIIb.