The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. (3); the N-terminal portion governs transport across the envelope, whereas the C-terminal portion directs translocation across the thylakoid membrane (4). In the case of LHCP, the mature apoprotein contains sufficient information for thylakoid targeting (5), although the specific signal has not yet been identified (6, 7). Studies of the early events of LHCP trafficking using isolated chloroplasts showed that LHCP is present as a soluble form in the stroma prior to integration into thylakoids (8). When integration was inhibited, this intermediate LHCP accumulated in the stroma as a larger complex (9). Formation of this complex, which we have designated the "transit complex," maintains LHCP solubility and integration competence (9). Both transit complex formation and LHCP integrationThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.into thylakoids have been reconstituted in vitro; both require a proteinaceous component(s) of the stroma (9, 10).The chaperone nature of this "stromal factor" led to the suggestions that heat shock proteins Cpn6O (11) and Hsp7O (12) were involved in LHCP integration. These molecular chaperones are abundant in the stroma (13,14), are able to bind polypeptides and release them upon ATP hydrolysis, and have been shown to be involved in mitochondrial protein import (15). However, immunoprecipitation experiments with antibodies against the two proteins failed to reveal the presence of Cpn6O and Hsp7O in the transit complex (9, 14). Furthermore, stromal extract depleted of Hsp7O still supported transit complex formation and LHCP integration, indicating that neither process requires this chaperone (14). The recent demonstration of a stromal GTP requirement (16) for LHCP integration now suggests the involvement of a guanine nucleotide-binding protein in this process. One likely candidate is 54CP, a chloroplast homologue of the 54-kDa protein of the mammalian signal recognition particle (SRP) (17). Protein transport across or integration into the endoplasmic reticulum (ER) is mediated by a GTP-dependent mechanism involving SRP (18). The 54-kDa polypeptide subunit of SRP (SRP54) plays a major role in SRP-dependent targeting. It binds guanine nucleotides as well as nascent polypeptides that are destined for ER transport or integration (19). Furthermore, SRP54 appears to be required for docking to the SRP receptor (20) and hydrolyzes the GTP required for dissociation of SRP from the receptor (21). In this report, we show that 54CP is bound to LHCP in the transit complex and is essential for LHCP integration into the thylakoid membrane. (p) were prepared by in vitro transcription and translation as described (7). Unlabeled pLHCP and pOE33 (33-kDa subunit of the oxygenevolving complex) were expressed in Escherichia coli and pur...

Rice prolamines are sequestered within the endoplasmic reticulum (ER) lumen even though they lack a lumenal retention signal. Immunochemical and biochemical data show that BiP, a protein that binds lumenal polypeptides, is localized on the surface of the aggregated prolamine protein bodies (PBs). BiP also forms complexes with nascent chains of prolamines in polyribosomes and with free prolamines with distinct adenosine triphosphate sensitivities. Thus, BiP retains prolamines in the lumen by facilitating their folding and assembly into PBs.

The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to 3 H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein.Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH.45 Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation.Many plants produce calcium (Ca) oxalate as crystalline deposits (Arnott and Pautard, 1970; Gallaher, 1975; Franceschi and Horner, 1980; Horner and Wagner, 1995; Webb, 1999; Nakata, 2003), which can account for greater than 85% of the dry weight of some plant organs. The formation of Ca oxalate is an essential process in many species, and more than 90% of tissue Ca can be tied up as this compound (Gallaher et al., 1975; Gallaher and Jones, 1976). Ca oxalate crystals often occur within the vacuole of crystal idioblasts (Foster, 1956), specialized cells that generally encompass less than 1% to 2% of the total cells of the Ca-accumulating tissue. Because Ca oxalate formation is the end result of a mechanism for controlling Ca at the tissue and organ levels in the plant (Zindler-Frank, 1975; Borchert, 1985; Franceschi, 1989 Franceschi, , 2001 DeSilva et al., 1996; Kuo-Huang and Zindler-Frank, 1998; Pennisi and McConnell, 2001; Zindler-Frank et al., 2001; Volk et al., 2002), cells producing the crystals are considered to be highcapacity Ca sinks. Because crystal idioblasts perform a unique complex function of importance to the general physiology of the plant, and they commonly occur as single cells scattered among other tissues, we have referred to them as single-celled organs (Kostman and Franceschi, 2000).Large amounts of Ca oxalate crystals can be formed...

The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.