Interaction of Actin and the Chloroplast Protein Import Apparatus

Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.Actin is a ubiquitous protein of eukaryotic cells. Actin microfilaments are formed from polymerization of actin monomers and are a major component of the cytoskeleton. In plant cells, actin microfilaments are arranged in longitudinal arrays of thick actin bundles with randomly oriented thin actin filaments extending from the bundles (1). Chloroplasts are either aligned along the actin bundles or closely associated with the fine filaments and are surrounded by baskets of actin microfilaments (1, 2). A direct interaction of chloroplasts with the actin cytoskeleton has been postulated to anchor chloroplasts at appropriate intracellular positions (3). Chloroplast movement depends on cytosolic actin filaments and is stimulated by high light intensity (4). A chloroplast envelope protein involved in blue light-dependent chloroplast repositioning has been identified by the analysis of the Arabidopsis chup1 (chloroplast unusual positioning 1) mutant, which was unable to relocate its chloroplasts under high light stimulation (5). CHUP1 is a protein exclusively targeted to the chloroplast outer envelope membrane that is essential for chloroplast anchorage to the plasma membrane (6). CHUP1 interacts with actin and profilin, a modulator of actin polymerization, and it may play a regulatory role in actin polymerization during chloroplast photo-relocation (7).The interaction of amyloplasts with the actin cytoskeleton has been implicated in gravity perception and signal transduction. Several models for the role of the actin cytoskeleton have been proposed (8), but the nature of the interaction is not known. However, disruption of the actin cytoskeleton enhanced sedimentation of amyloplasts and promoted gravitropism (9, 10), and a role for myosin has been proposed on the basis of inhibitor experiments (11).The actin cytoskeleton and myosin have also been implicated in plastid stromule movement. Stromules (stroma-filled tubules) are highly dynamic tubular structures extending from the surface of all plastid types (12, 13). Stromules are delimited by the inner and outer plastid envelope membranes, which are closely associated (for a review, see Refs. 12 and 13). Experiments with inhibitors of microfilament- and microtubule-based movement suggested that stromules move along actin microfilaments powered by the ATPase activity of myosin motors (14). Physical connection between the envelope membranes seems likely to be required to provide a means of coordinating the movement of the inner envelope membrane with the microfilament-associated outer envelope membrane. There is evidence for direct connection of the inner and outer envelope membranes at contact sites, which support protein translocation through the protein import apparatus (15, 16). This apparatus consists of two membrane protein complexes that associate to allow translocation of nucleus-encoded proteins from the cytoplasm to the interior stromal compartment (for a review, see 17). The translocon at the outer envelope membrane of chloroplasts (TOC complex)² mediates the initial recognition of preproteins and their translocation across the outer membrane (18). The translocon at the inner envelope membrane of chloroplasts (TIC complex) physically associates with the TOC complex and provides the membrane translocation channel for the inner membrane. In addition, the TOC and TIC complexes interact with a set of molecular chaperones, which assist the transfer of imported proteins (19–21).With the aim of identifying components involved in the interaction of the chloroplast envelope with the actin cytoskeleton, we have used actin co-immunoprecipitation and co-sedimentation experiments with detergent-solubilized pea chloroplast envelope membranes. Components of the TOC-TIC protein import apparatus have been identified by mass spectrometry and Western blotting, and a direct interaction of Escherichia coli-expressed Toc159 with actin was demonstrated by co-sedimentation. This interaction may have a so far unrecognized physiological role in chloroplast protein import.     

Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

蛋白质向叶绿体的转运

对近年来叶绿体蛋白质前导肽序列、叶绿体被膜中的蛋白质转运器、监护蛋白在蛋白转运过程中的作用、蛋白质导入叶绿体的途径、前体蛋白的加工的研究进展进行了介绍和评述     

A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH₂-proximal part of mature IEP110 (110N). The NH₂-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80–90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH₂-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.     

Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.     

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90). Here, we show that atToc90 supports accumulation of photosynthetic proteins in plastids, but is not required for import of several constitutive proteins. Part of atToc90 associates with the chloroplast surface in vivo and with the Toc-complex core components (atToc75 and atToc33) in vitro suggesting a function in chloroplast protein import similar to that of atToc159. As both proteins specifically contribute to the accumulation of photosynthetic proteins in chloroplasts they may be components of the same import pathway.     

The First Steps of Protein Import into Mitochondria

Protein import into mitochondria is initiated by the recognition and binding of precursor proteins by import components in the cytosol, on the mitochondrial surface, and in the mitochondrial outer membrane. Following their synthesis on cytoplasmic ribosomes, some precursor proteins interact with molecular chaperones in the cytosol which function in maintaining the precursor protein in an import-competent state and may also aid in the delivery of the precursor to the mitochondria. A multisubunit protein import receptor then recognises and binds precursor proteins before feeding them into the outer membrane import site. Some proteins are sorted from the import site into the outer membrane, but most precursor proteins travel through the outer membrane import site into the mitochondria, where the later steps of protein import take place.     

Molecular Characterization and Expression Analysis of Chloroplast Protein Import Components in Tomato (Solanum lycopersicum)

The translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and initial import into the organelle of thousands of nucleus-encoded proteins. These proteins are translated in the cytosol as precursor proteins with cleavable amino-terminal targeting sequences called transit peptides. The majority of the known Toc components that mediate chloroplast protein import were originally identified in pea, and more recently have been studied most extensively in Arabidopsis. With the completion of the tomato genome sequencing project, it is now possible to identify putative homologues of the chloroplast import components in tomato. In the work reported here, the Toc GTPase cDNAs from tomato were identified, cloned and analyzed. The analysis revealed that there are four Toc159 homologues (slToc159-1, -2, -3 and -4) and two Toc34 homologues (slToc34-1 and -2) in tomato, and it was shown that tomato Toc159 and Toc34 homologues share high sequence similarity with the comparable import apparatus components from Arabidopsis and pea. Thus, tomato is a valid model for further study of this system. The expression level of Toc complex components was also investigated in different tissues during tomato development. The two tomato Toc34 homologues are expressed at higher levels in non-photosynthetic tissues, whereas, the expression of two tomato Toc159 homologues, slToc159-1 and slToc159-4, were higher in photosynthetic tissues, and the expression patterns of slToc159-2 was not significantly different in photosynthetic and non-photosynthetic tissues, and slToc159-3 expression was limited to a few select tissues.     

Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.     

New Insights into the Protein Import Machinery of the Chloroplast's Outer Envelope

A large number of plastid localized proteins are post-translationally imported as precursor proteins from the cytosol into the organelle. Recognition and translocation is accomplished by a subset of chloroplast envelope proteins, which were identified by different but complementary methods. The o uter e nvelope p roteins OEP 86, OEP 75, OEP 70 (a heat shock cognate 70 homologue) and OEP 34 are clearly involved in the import event and can be isolated as one functionally active translocation unit. For three of these proteins cDNA clones have been very recently obtained, namely OEP 86, OEP 75 and OEP 34. OEP 86 seems to be a precursor protein receptor which could be regulated by GTP binding and ATP-dependent phosphorylation-dephosphorylation. OEP 75 is part of the translocation pore traversing the membrane in multiple β-sheets. OEP 34 is tightly associated with OEP 75. It represents a new type of GTP-binding protein which possesses endogenous GTPase activity. Multiple GTP binding and hydrolysis cycles as well as protein phosphorylation-dephosphorylation events might, therefore, regulate the interaction of a precursor protein with the translocation machinery of the outer envelope, making it very distinct from the mitochondrial outer membrane system. Further proteins of the inner envelope membrane, namely IEP 97 and IEP 36, have been implied to function in the translocation event. These recent data allow not only identification of the players in the game but also speculation about mechanisms and regulation of translocation.     

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

All mitochondrial tRNAs in Trypanosoma brucei derive from cytosolic tRNAs that are in part imported into mitochondria. Some trypanosomal tRNAs are thiolated in a compartment-specific manner. We have identified three proteins required for the thio modification of cytosolic tRNA^Gln, tRNA^Glu, and tRNA^Lys. RNA interference-mediated ablation of these proteins results in the cytosolic accumulation non-thio-modified tRNAs but does not increase their import. Moreover, in vitro import experiments showed that both thio-modified and non-thio-modified tRNA^Glu can efficiently be imported into mitochondria. These results indicate that unlike previously suggested the cytosol-specific thio modifications do not function as antideterminants for mitochondrial tRNA import. Consistent with these results we showed by using inducible expression of a tagged tRNA^Glu that it is mainly the thiolated form that is imported in vivo. Unexpectedly, the imported tRNA becomes dethiolated after import, which explains why the non-thiolated form is enriched in mitochondria. Finally, we have identified two genes required for thiolation of imported tRNA^Trp whose wobble nucleotide is subject to mitochondrial C to U editing. Interestingly, down-regulation of thiolation resulted in an increase of edited tRNA^Trp but did not affect growth.     

In vivo Studies on the Roles of Tic55-Related Proteins in Chloroplast Protein Import in Arabidopsis thaliana

The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.     

Silencing of Mammalian Sar1 Isoforms Reveals COPII‐Independent Protein Sorting and Transport

The Sar1 GTPase coordinates the assembly of coat protein complex‐II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER‐to‐Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII‐mediated transport in mammalian cells, we used small interfering RNA (siRNA)‐mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER‐to‐Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen‐I is inhibited. These data provide proof‐of‐principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII .     

A 1-Megadalton Translocation Complex Containing Tic20 and Tic21 Mediates Chloroplast Protein Import at the Inner Envelope Membrane

Chloroplast protein import is mediated by two hetero-oligomeric protein complexes, the Tic and Toc translocons, which are located in the inner and outer envelope membranes. At the inner membrane, many Tic components have been identified and characterized, but it remains unclear how these Tic proteins are organized to form a protein-conducting channel or whether a stable Tic core complex that binds translocating preproteins exists. Here, we report the identification of a 1-megadalton (MD) translocation complex as an intermediate during protein translocation across the inner membrane in Arabidopsis thaliana and pea (Pisum sativum). This complex can be detected by blue native PAGE using the mild detergent digitonin without any chemical cross-linkers. The preprotein arrested in the 1-MD complex can be chased into its fully translocated form after a subsequent incubation. While Tic20 and Tic21 appear to be involved in the 1-MD complex, Tic110, a well-characterized Tic component, exists as a distinct entity from the complex. Several lines of evidence suggest that the 1-MD complex functions in between the Toc and Tic110-containing complexes, most likely as a protein-conducting channel at the inner envelope.     

Apparent Inhibition of Chloroplast Protein Import by Cold Temperatures Is Due to Energetic Considerations Not Membrane Fluidity

The transport of proteins across virtually all types of biological membranes has been reported to be inhibited by low temperatures. Paradoxically, plants are able to acclimate to growth at temperatures below which protein import into chloroplasts is known to be blocked. In examining this incongruity, we made a number of unexpected observations. First, chloroplasts isolated from plants grown at 7/1[deg]C in light/dark and from plants grown at 25[deg]C were able to import proteins with the same efficiency over a temperature range from 5 to 21[deg]C, indicating that no functional adaptation had taken place in the protein import machinery of chloroplasts in these cold-grown plants. Second, chloroplasts from warm-grown plants were able to take up proteins at temperatures as low as 4[deg]C provided that they were illuminated. We determined that light mediates the import process at 5[deg]C by driving ATP synthesis in the stroma, the site of its utilization during protein transport. Direct measurement of the envelope phase transition temperature as well as the activity of the ATP/ADP translocator in the inner envelope membrane at 5 and 25[deg]C demonstrated that the cold block of protein import into chloroplasts observed in vitro is due primarily to energetic considerations and not to decreased membrane fluidity.     

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/γ-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, ΔGRT1 ΔGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ΔGRT1 ΔGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ΔGRT1 ΔGRT2 cells appear less adhesive than those from the wild type.In eukaryotes, the directional transport of lumenal proteins throughout the network of membrane-bound organelles depends on reversible assembly of multisubunit protein complexes in the cytoplasm. For example, the assembly of a localized clathrin coat at a cell''s surface facilitates both the concentration of specific transmembrane receptors together with their bound ligands at that site and the invagination and budding of the plasma membrane, resulting in endocytosis (18). Similarly, other cytosolic coats assemble and direct traffic at the endoplasmic reticulum (ER) and Golgi apparatus (4). For one protein trafficking pathway in eukaryotic cells, however, the determinative protein self-assembly occurs not in the cytoplasm but within the lumen of the secretory pathway itself. Dense core granules (DCGs) are secretory vesicles whose lumenal cargo consists of a condensed polypeptide aggregate. This cargo is secreted when the vesicles fuse with the plasma membrane in response to a specific extracellular stimulus, an event called regulated exocytosis. The aggregation of the cargo occurs progressively within the secretory pathway, beginning in the trans-Golgi network (TGN), and may be promoted by multiple factors including compartment-specific proton and calcium levels (23). Aggregation facilitates the vesicular storage of concentrated secretory proteins but also serves as a sorting mechanism to segregate DCG proteins from proteins that are secreted via other pathways. Evidence for this mechanism includes in vitro experiments showing that some proteins released via constitutive exocytosis remain soluble under TGN-like conditions that promote DCG protein aggregation (10). In vivo, sorting would result if aggregated and soluble proteins exit the TGN in different carriers. Importantly, there is no evidence that sorting of DCG proteins at the TGN requires assembly of cytosolic coat complexes.While aggregative sorting represents an attractively simple mechanism, relatively little is known about the structure or dynamic properties of the aggregates themselves. This is an interesting issue, as illustrated by several phenomena. First, aggregates in some cell types, like those formed by proinsulin in pancreatic β cells, can become reordered as protein crystals during a multistage process called granule maturation (13). Second, Aplysia bag cells can sort different subsets of DCG proteins into distinct granules, suggesting that aggregation can be finely regulated and that different aggregates have different properties in vivo (20). Both of these phenomena have also been observed within the DCGs of unicellular ciliates (3, 14). In addition, ciliate DCGs demonstrate another degree of subtlety in DCG formation because the granule cores in many of these organisms are divided into distinct domains (25). The domain organization indicates that DCG proteins in these cells can segregate from one another even as they are sorted to the same vesicular destination. While the structures of DCGs in many ciliates have been captured by electron microscopy, molecular studies have advanced in two species, Tetrahymena thermophila and Paramecium tetraurelia (30, 33).In many ciliates, the individual DCGs are organized in at least two distinct domains within the lumen. First, the bulk of the cargo is organized as a core crystal that expands, spring-like, upon exocytosis (28). This expansion can drive rapid extrusion of the DCG contents, which may be essential for hunting or defensive behaviors (17). In addition, many ciliate DCGs possess a single polarized tip structure that is involved in DCG docking to the plasma membrane and exocytic fusion (25). These tip structures are also filled with condensed, highly organized proteins, which appear by both genetic and morphological criteria to be different from proteins making up the expansible core (1, 21). The proteins that form the distinct domains are beginning to be identified and analyzed. Those that constitute the expansible springs are encoded by homologous families of genes named GRL (granule lattice) in Tetrahymena and tmp (trichocyst matrix) in Paramecium (11, 12, 15). Assembly of Grl proteins begins in the ER with formation of heterooligomers. This is an obligatory step, as shown by the fact that deletion of individual Grl proteins by targeted gene disruption resulted in the ER retention of remaining Grl proteins (12). Further assembly of Grl proteins to form a crystal occurs during DCG maturation and is accompanied by site-specific proprotein processing (34). Upon exocytosis, the expansion of the crystalline core is controlled by calcium binding to the fully processed Grl proteins (34).In addition to the GRL family-encoded proteins, 13 other lumenal DCG proteins have been putatively or definitively identified in Tetrahymena, and homologous proteins are predicted in the Paramecium genome (6). The entire set belongs to a gene family that is defined by a carboxy-terminal β/γ-crystallin domain, which may function as a DCG-targeting motif (16). Studies of two different members of this family in Tetrahymena, IGR1 (induced during granule regeneration 1) and GRT1 (granule tip 1), suggested that these proteins are functionally distinct from the spring-forming Grl proteins. First, whereas gene disruption of any of the highly transcribed GRL genes resulted in grossly aberrant spring formation, no such defect was seen upon disruption of IGR1 (16). However, this could be explained by the fact that IGR1 encodes a relatively low-abundance protein in DCGs, and furthermore its function could be redundant with that of the highly related gene, IGR2.The second protein in the β/γ-crystallin domain family that has been investigated is the 80-kDa product of the GRT1 gene. Grt1p was first detected as one of the most abundant DCG components released during exocytosis (32). Biochemical analysis showed that Grt1p differs in its solubility from the Grl proteins and also that it is packaged intact in DCGs rather than undergoing proteolytic processing (31). Since processing is essential for Grl protein assembly and function, this difference appears highly significant. Second, Grt1p accumulates at a single pole of each DCG, corresponding to the tip of the organelle that docks and then fuses with the plasma membrane (5). Two Mendelian mutants with defects in DCG maturation show delocalized Grt1p, and these mutant DCGs can dock but do not appear to undergo exocytosis (5). These results suggested that Grt1p might be involved in forming a DCG tip domain that interacted with the plasma membrane.We have now investigated the trafficking and function of Grt1p. Our data provide both direct biochemical and cell-biological evidence that Grt1p and Grl proteins form distinct complexes during DCG biogenesis in Tetrahymena. Together with earlier results, our experiments provide genetic evidence that Grl and Grt complexes can be independently trafficked to DCGs. Cells lacking GRT1, together with the closely related GRT2, still show rapid and efficient release of DCG contents upon stimulation with secretagogues, but the released DCG contents are subtly different from those of the wild type, suggesting that Grt1p may primarily serve a postexocytic function.     

Alternative Processing of Arabidopsis Hsp70 Precursors during Protein Import into Chloroplasts

During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.     

The Molecular Basis for Distinct Pathways for Protein Import into Arabidopsis Chloroplasts

The translocons at the outer envelope membrane of chloroplasts (TOCs) initiate the import of thousands of nucleus-encoded proteins into the organelle. The identification of structurally and functionally distinct TOC complexes has led to the hypothesis that the translocons constitute different import pathways that are required to coordinate the import of sets of proteins whose expression varies in response to organelle biogenesis and physiological adaptation. To test this hypothesis, we examined the molecular basis for distinct TOC pathways by analyzing the functional diversification among the Toc159 family of TOC receptors. We demonstrate that the N-terminal A-domains of the Toc159 receptors regulate their selectivity for preprotein binding. Furthermore, the in vivo function of the two major Toc159 family members (atToc159 and atToc132) can be largely switched by swapping their A-domains in transgenic Arabidopsis thaliana. On the basis of these results, we propose that the A-domains of the Toc159 receptors are major determinants of distinct pathways for protein import into chloroplasts.     

Retromer-Mediated Protein Sorting and Vesicular Trafficking

Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking,developmental signaling and lysosome biogenesis.Through its interaction with the Rab GTPases and their effectors,membrane lipids,molecular motors,the endocytic machinery and actin nucleation promoting factors,retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network(TGN) and the plasma membrane.In this review.I highlight recent progress in the understanding of relromer-medialed protein sorting and vesicle trafficking and discuss how retromer contributes to a diverse set of developmental,physiological and pathological processes.