Many of the proteins in the chloroplast envelope play an important role in facilitating the biochemical and transport processes of the compartment. For the transport of proteins into the chloroplast, we have recently identified at least three different envelope proteins (Com44/Cim44, Com70, and Cim97) in close physical proximity to a partially translocated chimeric precursor protein (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264 -32271). In this study we report the characterization of a cDNA clone encoding a member of the Com44/Cim44 envelope proteins. The combined data from nucleotide sequencing, and RNA and protein blot analyses indicate the existence of multiple forms of the 44-kDa envelope protein. Depending on the plant species examined, immunologically-related protein bands with molecular masses of 42 to 46 kDa were observed. Organelle subfractionation, protease treatment, and immunomicroscopic studies together provide an indication that the immunologically-related proteins may be present in both the outer and inner envelope membranes. Co-migration of the product synthesized from the cDNA insert with a 44-kDa immunoreactive band of the chloroplast envelope, and the in vitro import results, together suggest that the in vitro synthesized 44-kDa protein is targeted to the envelope membrane without any further processing.Chloroplast envelope proteins play a major role in modulating the vectorial flow of molecules across the membrane, including large proteinaceous entities. The import of proteins into the chloroplast is a complex process requiring the close collaboration of both the outer envelope and the inner envelope membranes. Evidence for the possible existence of two distinct protein import complexes, one in each envelope membrane, is beginning to emerge from a number of recent investigations (Waegemann and Soll, 1991;Soll and Waegemann, 1992;Schnell and Blobel, 1993;Alefson et al., 1994; Schell et al., 1994;Kessler et al., 1994;Wu et al., 1994). An important step in the characterization of the protein translocating complexes is the identification of the components involved. The identification of outer and inner envelope polypeptides of these protein translocating complexes has been achieved using a variety of strategies (Cornwall and Keegstra, 1987;Kaderbhai et al., 1988;Pain et al., 1988; Schnell et al., 1990aSchnell et al., , 1994Hinz and Flugge, 1988;Soll and Waegemann, 1992;Waegemann et al., 1990;Perry and Keegstra, 1994;Alefson et al., 1994;Wu et al., 1994;Hirsch et al., 1994;Gray and Row, 1995). So far these studies collectively indicate that envelope proteins with molecular masses of 30, 34, 36, 44, 45, 51, 66, 70, 75, 86, 97, and 100 kDa may be possible constituents of the chloroplast protein import apparatus; however, it is not obvious from the existing data whether some of the predicted similar sized components are identical to each other.The complex nature of protein translocation mechanisms observed in other membranous systems, such as the mitochondrion and the endoplas...

Lhcb1-2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1-2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25 degrees C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4 degrees C for 4 h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1-2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.

The transport of proteins across the plastid envelope involves a host of proteinaceous components that attend to varying structural needs of the process. This study focuses on interactions between two select forms (designated Tic40 and Toc36) of the Tic40-related components and different structural versions of the Oee1 precursor to dissect the components' mode of operation. Interaction profiling revealed several features pertaining to how Tic40-related components might work during the transport process. The main operational features revealed are: (1) Tic40 interacts preferentially with Oee1 precursors containing only the plastid-targeting domain, (2) Toc36 interacts preferentially with Oee1 precursors containing both plastid- and thylakoid lumen-targeting domains, (3)�carboxyl truncations to either the entire Oee1 precursor or Toc36 affect interactions negatively, and (4) the general reduction of Tic40-related protein levels in transgenic plants appears to exert a greater negative impact on endogenous Oee1 levels than the other proteins assessed, a trend that corroborates the findings of the protein interaction experiments.

The transport of proteins into the plastid is a process that faces changing cellular needs such as the situation found in different plant organs or developing tissues. The plastid translocon must therefore be responsive to the changing cell environment to deliver efficiently different arrays of structurally diverse proteins. Although the Tic40-related envelope proteins appear to be translocon components designed to address the varying needs of protein translocation, details of their involvement remain elusive. This study was thus designed to combine plant-based experiments and yeast mitochondrion-based approaches for unveiling clues related to how the Tic40 components may behave during the protein translocation process. The main findings related to how Tic40 proteins may work are: 1) natural fluctuations are apparent in developing tissues, in different organs of the same plant, and in different species; 2) transgenic Arabidopsis seedlings can tolerate functionally a wide range of variations in Tic40 levels, from partial suppression to excessive production; 3) the Tic40 proteins themselves exhibit configurational changes in their association with yeast mitochondria in response to different carbon sources; 4) the presence of Tic40 proteins in yeast mitochondria influences regulatory aspects of the mitochondrial translocon; and 5) the Tic40 proteins associate with mitochondrial translocon components involved in regulatory-like events. The combined data provide evidence that Tic40 proteins possess modulating capabilities.Plastids are diverse in structure and function and occupy key roles in a variety of biosynthetic activities that take place in the ever changing environment of a plant cell. The role of plastids requires frequent adjustments to accommodate varying cellular needs such as those occurring in different organs, during development, during adaptation to external stimuli, or even a combination of needs. The adjustments required can be relatively subtle or distinct. The ability of the plastid to address such needs is dependent on its compositional status, e.g. chloroplasts versus leucoplasts. Although it is well established that changes to plastidial structure and function are governed predominantly by mechanisms that control gene expression (nuclear and organellar), there is growing evidence that the protein transport process contributes additionally to certain facets of these regulatory mechanisms. The involvement of protein transport in the various mechanisms of plastidial change is not unfounded because most of the plastid proteins involved are made as larger precursors before incorporation into organelles. The protein transport machinery of the plastid envelope (the plastid translocon) must therefore be accommodative in its ability to handle in an efficient manner a diverse, ever changing array of proteins. The proteins being handled range from different proteins to structural variations of the same protein.The plastid translocon is also afforded a position where additional "modulating" mechanisms can b...

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