Translation In Wheat Germ Extract Research Articles

-1 Programmed ribosomal frameshifting in Class 2 umbravirus-like RNAs uses multiple long-distance interactions to shift between active and inactive structures and destabilize the frameshift stimulating element.

Plus-strand RNA viruses frequently employ -1 programmed ribosomal frameshifting (-1 PRF) to maximize their coding capacity. Ribosomes can frameshift at a slippery sequence if progression is impeded by a frameshift stimulating element (FSE), which is generally a stable, complex, dynamic structure with multiple conformations that contribute to the efficiency of -1 PRF. AsFSE are usually analyzed separatefrom the viral genome, little is known about cis-acting long-distance interactions. Using full-length genomic RNA of umbravirus-like (ula)RNA citrus yellow vein associated virus (CY1) and translation in wheat germ extracts, sixtertiary interactions were found associated with the CY1 FSE that span nearly three-quartersof the 2.7 kb genomic RNA. All sixtertiary interactions are conserved in other Class 2 ulaRNAs and two are conserved in all ulaRNAs. Two sets of interactions comprise local and distal pseudoknots that involve overlapping FSE nucleotides and thus are structurally incompatible, suggesting that Class 2 FSEs assume multiple conformations. Importantly, two long-distance interactions connect with sequences on opposite sides of the critical FSE central stem, which would unzip the stem and destabilize the FSE. These latter interactions could allow a frameshifting ribosome to translate through a structurally disrupted upstream FSE that no longer blocks ribosome progression.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Novel 3' Proximal Replication Elements in Umbravirus Genomes.

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.

Mapping of sequences in the 5' region and 3' UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

A Detailed Protocol for Preparing Millimeter-sized Supergiant Liposomes that Permit Efficient Eukaryotic Cell-free Translation in the Interior.

Liposomes have been used as a pseudo cell membrane for encapsulating biomolecules and creating an artificial cell in the interior where biochemical reactions can occur. Among the several methods used to prepare biomolecule-encapsulating liposomes, the spontaneous emulsion transfer method is superior to others in that it allows us to readily prepare relatively large liposomes whose sizes are controlled (from micrometer- to millimeter-sized liposomes) without special equipment. However, conventional protocols for this method require liposomes to contain a considerably high concentration of sucrose (high-density solute), which severely inhibits gene expression, one of the most important biochemical reactions. Thus, we optimized the preparation conditions to develop a wheat germ extract (WGE)-based protocol that requires a much lower concentration of sucrose and has almost no effect on eukaryotic cell-free translation. Our protocol allows us to successfully prepare millimeter-sized, moderately stable, WGE-encapsulating liposomes in which WGE translation takes place efficiently. Since a broad range of genes derived from various types of organisms can be efficiently translated in a WGE-based translation system, liposomes prepared using our protocol would be useful as a versatile research tool for artificial cells.

The 3' Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems.

Many plant viral RNA genomes lack a 5′ cap, and instead are translated via a cap-independent translation element (CITE) in the 3′ untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host’s translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses.

Position-dependent effects of regioisomeric methylated adenine and guanine ribonucleosides on translation.

Reversible methylation of the N6 or N1 position of adenine in RNA has recently been shown to play significant roles in regulating the functions of RNA. RNA can also be alkylated upon exposure to endogenous and exogenous alkylating agents. Here we examined how regio-specific methylation at the hydrogen bonding edge of adenine and guanine in mRNA affects translation. When situated at the third codon position, the methylated nucleosides did not compromise the speed or accuracy of translation under most circumstances. When located at the first or second codon position, N1-methyladenosine (m1A) and m1G constituted robust blocks to both Escherichia coli and wheat germ extract translation systems, whereas N2-methylguanosine (m2G) moderately impeded translation. While m1A, m2G and N6-methyladenosine (m6A) did not perturb translational fidelity, O6-methylguanosine (m6G) at the first and second codon positions was strongly and moderately miscoding, respectively, and it was decoded as an adenosine in both systems. The effects of methylated ribonucleosides on translation could be attributed to the methylation-elicited alterations in base pairing properties of the nucleobases, and the mechanisms of ribosomal decoding contributed to the position-dependent effects. Together, our study afforded important new knowledge about the modulation of translation by methylation of purine nucleobases in mRNA.

Translational readthrough in Tobacco necrosis virus-D

The plus-strand RNA genome of Tobacco necrosis virus-D (TNV-D) expresses its polymerase via translational readthrough. The RNA signals involved in this readthrough process were characterized in vitro using a wheat germ extract translation system and in vivo via protoplast infections. The results indicate that (i) TNV-D requires a long-range RNA-RNA interaction between an extended stem-loop (SL) structure proximal to the readthrough site and a sequence in the 3'-untranslated region of its genome; (ii) stability of the extended SL structure is important for its function; (iii) TNV-D readthrough elements are compatible with UAG and UGA, but not UAA; (iv) a readthrough defect can be rescued by a heterologous readthrough element in vitro, but not in vivo; and (v) readthrough elements can also mediate translational frameshifting. These results provide new information on determinants of readthrough in TNV-D and further support the concept of a common general mechanism for readthrough in Tombusviridae.

RNA complexity during seed formation and germination.

ABSTRACT The sequence composition and quantity of cotton cotyledon mRNAs present after 24 h of germination were evaluated by physical techniques, in vitro translation in wheat germ extracts, and hybridization with complementary DNA. The results indicate that most of the 25,000 individual mRNAs exist in both the poly(A)plus mRNA and the poly(A)minus mRNA populations in similar concentrations. Polysomal and nonpolysomal mRNA populations also appear to be virtually identical. The cotyledon mRNAs present during cotyledon development were compared by hybridization of poly(A)plus mRNA from each of three stages with DNA complementary to RNAs present in the other stages. Dramatic changes in the concentration of most of the abundant mRNA sequences and many of the less abundant mRNA sequences occur during cotyledon development. Most of the about 25,000 rare mRNA sequences appear to be common to all three stage mRNAs. A summary is presented of the mRNA sequences in other plant issues, as reported from other laborato...

Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency

BackgroundWhole plants or plant cell cultures can serve as low cost bioreactors to produce massive amounts of a specific protein for pharmacological or industrial use. To maximize protein expression, translation of mRNA must be optimized. Many plant viral RNAs harbor extremely efficient translation enhancers. However, few of these different translation elements have been compared side-by-side. Thus, it is unclear which are the most efficient translation enhancers. Here, we compare the effects of untranslated regions (UTRs) containing translation elements from six plant viruses on translation in wheat germ extract and in monocotyledenous and dicotyledenous plant cells.ResultsThe highest expressing uncapped mRNAs contained viral UTRs harboring Barley yellow dwarf virus (BYDV)-like cap-independent translation elements (BTEs). The BYDV BTE conferred the most efficient translation of a luciferase reporter in wheat germ extract and oat protoplasts, while uncapped mRNA containing the BTE from Tobacco necrosis virus-D translated most efficiently in tobacco cells. Capped mRNA containing the Tobacco mosaic virus omega sequence was the most efficient mRNA in tobacco cells. UTRs from Satellite tobacco necrosis virus, Tomato bushy stunt virus, and Crucifer-infecting tobamovirus (crTMV) did not stimulate translation efficiently. mRNA with the crTMV 5′ UTR was unstable in tobacco protoplasts.ConclusionsBTEs confer the highest levels of translation of uncapped mRNAs in vitro and in vivo, while the capped omega sequence is most efficient in tobacco cells. These results provide a basis for understanding mechanisms of translation enhancement, and for maximizing protein synthesis in cell-free systems, transgenic plants, or in viral expression vectors.

Improvement of in vitro-transcribed amber suppressor tRNAs toward higher suppression efficiency in wheat germ extract

In vitro-transcribed, unmodified, and non-aminoacylated amber suppressor tRNAs that are recognized by natural aminoacyl-tRNA synthetase were improved toward higher suppression efficiency in batch-mode cell-free translation in wheat germ extract. The suppression efficiency of the suppressor obtained through four sequence optimization steps (anticodon alteration of natural tRNAs (the first generation); chimerization of the efficient suppressors in the first generation; investigation and optimization of the effective parts in the second generation; combination of the optimized parts in the third generation) and by the terminal tuning was approximately 60%, which was 2.4-fold higher than that of the best suppressor in the first generation. In addition, an eRF1 aptamer further increased the efficiency up to 85%. This highly efficient suppression system also functioned well in a dialysis-based large-scale protein synthesis.

Effects of Viral Genome‐Linked Protein on Depurination of Viral RNA by Pokeweed Antiviral Protein

Pokeweed antiviral protein (PAP) is a ribosome inactivating protein and is an N‐glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA and arrests protein synthesis at the translocation step. PAP, a potent antiviral agent against many plant, animal, and human viruses, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNA depurinating them at sites downstream of the cap structure. To understand the molecular basis of PAP's antiviral activity, using fluorimetric methods, we examine interactions occurring between PAP and Potyvirus genome linked protein (VPg) that is shown to perform as a cap analog in viral cap‐independent translation of RNA bearing internal ribosome entry site (IRES). VPg interacts with cap‐binding proteins, stimulates translation of uncapped IRES‐containing RNA, and inhibits capped RNA translation in wheat germ extract. We have characterized interaction of PAP with VPg, and structured RNA derived from tobacco etch virus (TEV). When VPg complexes with PAP, the affinity for uncapped TEV RNA tripled compared with PAP alone, whereas affinity of PAP for capped RNA was not altered significantly. This selective preference of PAP for uncapped RNA in the presence of VPg stimulates the idea of creating a chimeric PAP‐VPg mutant that possesses selectivity to depurinate uncapped viral RNA by directing it to viral IRES.

Cylindrical inclusion protein of potato virus A is associated with a subpopulation of particles isolated from infected plants

Potato virus A (PVA) particles were purified by centrifugation through a 30 % sucrose cushion and the pellet (P1) was resuspended and sedimented through a 5-40 % sucrose gradient. The gradient separation resulted in two different virus particle populations: a virus fraction (F) that formed a band in the gradient and one that formed a pellet (P2) at the bottom of the gradient. All three preparations contained infectious particles that retained their integrity when visualized by electron microscopy (EM). Western blotting of the P1 particles revealed that the viral RNA helicase, cylindrical inclusion protein (CI), co-purified with virus particles. This result was confirmed with co-immunoprecipitation experiments. CI was detected in P2 particle preparations, whereas F particles were devoid of detectable amounts of CI. ATPase activity was detected in all three preparations with the greatest amount in P2. Results from immunogold-labelling EM experiments suggested that a fraction of the CI present in the preparations was localized to one end of the virion. Atomic force microscopy (AFM) studies showed that P1 and P2 contained intact particles, some of which had a protruding tip structure at one end, whilst F virions were less stable and mostly appeared as beaded structures under the conditions of AFM. The RNA of the particles in F was translated five to ten times more efficiently than RNA from P2 particles when these preparations were subjected to translation in wheat-germ extracts. The results are discussed in the context of a model for CI-mediated functions.

RAP55, a Cytoplasmic mRNP Component, Represses Translation in Xenopus Oocytes

mRNAs in eukaryotic cells are presumed to always associate with a set of proteins to form mRNPs. In Xenopus oocytes, a large pool of maternal mRNAs is masked from the translational apparatus as storage mRNPs. Here we identified Xenopus RAP55 (xRAP55) as a component of RNPs that associate with FRGY2, the principal component of maternal mRNPs. RAP55 is a member of the Scd6 or Lsm14 family. RAP55 localized to cytoplasmic foci in Xenopus oocytes and the processing bodies (P-bodies) in cultured human cells: in the latter cells, RAP55 is an essential constituent of the P-bodies. We isolated xRAP55-containing complexes from Xenopus oocytes and identified xRAP55-associated proteins, including a DEAD-box protein, Xp54, and a protein arginine methyltransferase, PRMT1. Recombinant xRAP55 repressed translation, together with Xp54, in an in vitro translation system. In addition, xRAP55 repressed translation in oocytes when tethered with a reporter mRNA. Domain analyses revealed that the N-terminal region of RAP55, including the Lsm domain, is important for the localization to P-bodies and translational repression. Taken together, our results suggest that xRAP55 is involved in translational repression of mRNA as a component of storage mRNPs.

The -4 Phenylalanine Is Required for Substrate Ubiquitination Catalyzed by HECT Ubiquitin Ligases

The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.

Identification of the Subgenomic mRNAs That Encode 6-kDa Movement Protein and Hsp70 Homolog of Beet Yellows Virus

A tandem arrangement of the genes encoding the ∼6-kDa hydrophobic protein (p6) and Hsp70 homolog (Hsp70h) is conserved among the members of the Closterovirus genus. It was not known, however, if these movement proteins are expressed from one or two subgenomic (sg) RNAs. Here we employ RNA ligase-mediated RACE to show that the Beet yellows virus (BYV), a prototype Closterovius, produces separate sgRNAs encoding p6 and Hsp70h. This result is further supported by generation of the recombinant BYV in which the truncated variants of these sgRNAs are resolved by Northern analysis. The 5′-termini of the p6 and Hsp70h sgRNAs are localized to BYV nucleotides G-9402 and A-9467, respectively. Each of the sgRNAs was generated in vitro and found to direct the expected product upon translation in wheat germ extract. Inactivation of the first start codons in these sgRNAs abolished translation of the each product. The polyclonal antibodies raised to synthetic C-terminal peptides of p6 and Hsp70h specifically recognized corresponding translation products, as well as p6 and Hsp70h produced in BYV-infected plants. Taken together with the previous work, our data demonstrate that expression of the BYV genome involves the formation of as many as seven sgRNAs.

The 5' and 3' extremities of the satellite tobacco necrosis virus translational enhancer domain contribute differentially to stimulation of translation.

The translational enhancer domain (TED) of satellite tobacco necrosis virus (STNV) RNA stimulates translation of uncapped RNAs autonomously. Here we set out to identify the 5' and 3' extremities of TED and features of these sequences with respect to translation. We found that both in wheat germ extract and in tobacco protoplasts, the 5' border is confined to 3 nt. Mutational analysis revealed that the autonomous function of TED is sensitive to 5' flanking sequences. At the 3' end of TED, 23 nt have a cumulative, quantitative effect on translation in wheat germ extract, whereas in tobacco protoplasts, the most 3' 14 nt of these 23 nt do not enhance translation. The 5' and 3' sequence requirements triggered the development of a new secondary structure model. In this model, TED folds into a phylogenetically conserved stem-loop structure in which the essential 5' nucleotides base-pair with the 3' nucleotides that stimulate translation both in vitro and in vivo. Importantly, the 14 3' nucleotides in TED that stimulate translation in the wheat germ extract only do not require the predicted base-pairing in order to function. The discrepancy between in vitro and in vivo sequence requirements thus correlates with potential base-pairing requirements, opening the possibility that TED contains two functional domains.

In vitro-translated diphtheria toxin A chain inhibits translation in wheat germ extracts: analysis of biologically active, caspase-3-resistant diphtheria toxin mutants

The diphtheria toxin A chain (DTA) is a potent cytocidal agent that inactivates elongation factor 2. This activity of DTA inhibits protein synthesis and rapidly leads to cell death through apoptosis. In this paper, we have developed a simple in vitro assay for DTA activity in which in vitro-translated DTA is used to inhibit the translation of proteins in wheat germ extracts. Inhibition of translation by DTA is dependent on cofactor NAD +, and the analysis of an attenuated DTA mutant indicates that this in vitro assay accurately reflects the in vivo activity of DTA. We have also identified aspartic acid at residue 8 (Asp-8) of DTA as a site of cleavage by the cell-death protease caspase-3. Cleavage of DTA by caspase-3 inactivates its ability to inhibit translation in wheat germ extracts. Conservative mutations at Asp-8 render DTA resistant to cleavage by caspase-3, but only slightly affect the ability of DTA to inhibit translation in vitro. Moreover, caspase-3-resistant DTA mutants are toxic in cells in tissue culture. The in vitro assay that we describe here will be useful for the rapid analysis of DTA activity and the development of DTA mutants with altered biological properties that may be of therapeutic value. Lastly, these studies serve as a prototype for the creation of caspase-resistant effector molecules.

Membrane insertion, processing, and topology of cystic fibrosis transmembrane conductance regulator (CFTR) in microsomal membranes.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated C1(-) channel. Malfunction of CFTR causes cystic fibrosis (CF). CFTR belongs to an ATP-binding cassette (ABC) transporter superfamily which includes P-glycoprotein (Pgp), the molecule that is responsible for multidrug resistance in cancer cells. P-glycoprotein molecules have been suggested to have more than one topology and function. In this study, we analysed the early stages of membrane insertion, processing, and topology of human CFTR using rabbit reticulocyte lysate and wheat germ extract translation systems supplemented with canine pancreatic microsomal membranes. Our results suggest that CFTR contains an uncleavable signal sequence and its membrane targeting and insertion may depend on the signal recognition particle (SRP) and SRP receptor. The topology of CFTR in microsomal membranes is the same as the one predicted based on hydropathy plot analysis. These results, together with our previous findings on Pgp, indicate that (1) the topologies of mammalian ABC transporters can be dissected and studied using protein fusion chimeras in a cell-tree system; and (2) the membrane targeting and insertion of CFTR and Pgp may take the same pathway, i.e., the SRP-dependent pathway, but the membrane folding mechanism of these two proteins in microsomal membranes is probably different.

PSII-T, a New Nuclear Encoded Lumenal Protein from Photosystem II: TARGETING AND PROCESSING IN ISOLATED CHLOROPLASTS

An intronless nuclear gene, psbT, isolated from cotton, encodes a putative 11-kDa protein (PSII-T) highly homologous in its C terminus to the N terminus of the partially sequenced PSII-T protein from spinach photosystem II. Analysis of the deduced amino acid sequence of cotton PSII-T revealed the presence of potential chloroplast stroma and thylakoid targeting domains and a putative mature PSII protein of 3.0 kDa, composed of only 28 amino acid residues. The cotton PSII-T 11-kDa precursor was synthesized in vitro in a wheat germ extract translation system, and the translation product was used in assays for protein imports into isolated pea chloroplasts. It was shown that the cotton PSII-T precursor was imported into the chloroplasts, processed to a mature form of approximately 3.0 kDa, agreeing with the predicted size from amino acid sequence analysis, and localized to the lumenal side of the thylakoid membrane, thus defining a new nuclear encoded lumenal protein and the smallest polypeptide of PSII reported to date. Processing of the PSII-T precursor occurred in two steps and involved the formation of a stromal intermediate of approximately 7.5 kDa, as predicted from primary structure analysis.