The Early Stage of Chloroplast Protein Import Involves Com70

Abstract

The chloroplast envelope protein Com70 is a hsp70 homolog identified recently as a component of the protein translocation apparatus. The stage of protein import involving Com70 was determined by examining the nature of the association of Com70 with the envelope and its interaction with translocating proteins. Com70 is accessible to thermolysin, but its association with the envelope could not be disrupted by stringent washes. In light of the external membrane-bound location, the involvement of Com70 at the early stage of protein translocation was investigated using a combination of in vitro binding assays, chemical cross-linking, and coimmunoprecipitation. The results provide evidence that Com70 is in close physical proximity to different types of chloroplast protein precursors under conditions supporting binding rather than complete translocation. The formation of cross-linked complexes is dependent on the presence of a typical plastid transit signal and proteaseaccessible outer envelope components. The close proximity of Com70 and the translocating protein occurs while the protein is still exposed to the cytosol.The mechanism for importing chloroplast protein precursors into the compartment requires energy and is facilitated by specific envelope proteins (1). Recent studies revealed that envelope proteins with sizes of 34, 36, 44, 70 (at least two such related proteins), 75, 86, and 97 kDa are in close physical proximity to translocating proteins (1, 2). Although the role of each component remains to be elucidated, the outer envelope location of the 34-, 70-, 75-, and 86-kDa proteins places their involvement in the earlier part of the import pathway. The combination of GTP-binding domains in the 34-and 86-kDa proteins and the inhibition of precursor binding with nonhydrolyzable GTP analogs suggests that these two components may be involved in the energy-requiring aspect of the binding step (3-5). The 75-kDa protein is hypothesized, based on its predicted characteristics, to be a candidate for the import channel (6, 7). The 70-kDa components are related forms of the heat shock proteins (hsp70s) and may thus act similarly as chaperones/unfoldases (6,8,9).The importance of hsp70 in protein transport is exemplified by their widespread involvement in translocation mechanisms of the cytosol and/or the interior of different organelles (10 -12). Chloroplast protein import also appears to involve multiple hsp70-containing sites (cytosol, stroma, and envelope) (6,(13)(14)(15)(16). Since the chloroplast protein import machinery contains hsp70-related components at different locales within the outer envelope, external (Com70) versus internal (Iap70 and Hsc70) (6, 14, 17) it would be important to assess the individual contribution of each different hsp70-containing locale and the stage of its involvement. In this study we assessed further the role of Com70 in protein import by first examining the nature of the Com70 association with the outer envelope. In light of the strong association of Com70 with the cytosolic face of t...

Arabidopsis Genes Encoding Components of the Chloroplastic Protein Import Apparatus

“…Although no stromal Hsp70 proteins have been found to interact with import complexes (Akita et al, 1997;Nielsen et al, 1997), there is evidence to suggest that Hsp70 molecules do bind to precursor proteins before and/or during envelope translocation (Schnell et al, 1994;Wu et al, 1994;Kourtz and Ko, 1997;Ivey et al, 2000;May and Soll, 2000). Furthermore, an outer membrane-associated Hsp70 protein, which faces the intermembrane space of the chloroplast envelope, is believed to interact with precursor proteins as they move between the outer and inner membrane translocons (Marshall et al, 1990;Schnell et al, 1994).…”

Heat Shock Protein Cognate 70-4 and an E3 Ubiquitin Ligase, CHIP, Mediate Plastid-Destined Precursor Degradation through the Ubiquitin-26S Proteasome System inArabidopsis

“…The high levels of Hsc70-4 may play a role in either enhancing protein import into chloroplasts, as observed with spinach (Spinacia oleracea) chloroplast outer membrane-associated SCE70 (Com70) and Hsp70 in the guidance complex (Ko et al, 1992;Kourtz and Ko, 1997;Jarvis and Soll, 2002), or degradation of unimported precursors, as observed in protein quality control in animal and yeast cells (Figure 1; Esser et al, 2004). We examined the effect of high Hsc70-4 levels on protein import into chloroplasts using various chloroplast-targeted GFP proteins (RbcS-tp: GFP, Cab-tp:GFP [GFP fused to Cab TP], and RA-tp:GFP [GFP fused to ribulose-1,5-bisphosphate carboxylase/oxygenase activase TP]; see Supplemental Figure 4 online).…”

The chlorate‐resistant and photomorphogenesis‐defective mutant cr88 encodes a chloroplast‐targeted HSP90

“…Three HSP70s exist in the chloroplast. Two of them, LAP70 and COM70, have been identi®ed as components of the import machinery (Kourtz and Ko, 1997;Schnell et al, 1994). Stromal HSP70 proteins, which are not part of import machinery (Nielsen et al, 1997), have been found to interact with newly imported ferredoxin-NADP reductase (Tsugeki and Nishimura, 1993) and the Reiske iron-sulfur protein (Madueno et al, 1993).…”