Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane

Abstract

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

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

“…These results are supported by the crystal structure of the pea Toc34 homodimer identifying sequence motifs conserved in both atToc33 and atToc159 which are required for dimerization and chloroplast targeting of soluble atToc159 (Sun et al, 2002;Weibel et al, 2003). These findings suggest that atToc159 and its homologues may function as soluble factors in chloroplast protein import (Hiltbrunner et al, 2001b;Bauer et al, 2002;Smith et al, 2002). The Toc GTPase-system is strikingly analogous to the SRP system (which also employs a targeting mechanism based on two homotypic GTP-binding proteins) (Keenan et al, 2001) as well as to the Sec-system: SecA is present as soluble protein in the cytosol but also as a membrane-attached protein in the outer bacterial membrane and may drive protein translocation across the periplasmic membrane (Economou and Wickner, 1994) in an ATP-dependent, sewing machine-type fashion.…”

“…pET21d-atToc159 has been described (Bauer et al, 2000). pPCR-ScriptatToc159 1-731 , which contains the A-domain of atToc159 under the control of the T7 promoter, has been described (Bauer et al, 2002). The constructs described above were used for in vitro synthesis of [ 35 S]methionine labeled proteins in a reticulocyte-based coupled transcription/translation system (Promega, Madison WI, USA).…”

“…The construct for transient expression of GFPatToc159GM as well as pSH11 and pCL60 have been described (Bauer et al, 2002). The construct for transient expression of GFP-atToc90 was obtained as follows: A DNA fragment encoding atToc90 was amplified from EST-clone H4C12T7 using 5¢ and 3¢ primers including NotI sites (5¢ primer: 5¢-ATA AGA ATG CGG CCG CGA TGA AAG GCT TCA AAG ACT G-3¢; 3¢ primer: 5¢-ATA AGA ATG CGG CCG CTT AGG AAA CGA GAA AAT TCA CA-3¢).…”

“…DNA extraction and diagnostic PCR was done as described (Bauer et al, 2002). Plants containing a T-DNA insertion in TOC90 were identified by PCR using the primer set 5¢-ATA AGA ATG CGG CCG CGA TGA AAG GCT TCA AAG ACT G-3¢/5¢-AGA TAG GCT GAC ACG CCA AG-3¢.…”

“…Recently, we have discovered that atToc159 also exists in an abundant soluble form, partitioning between the outer chloroplast membrane and the cytosol (Hiltbrunner et al, 2001b). Cytosolic atToc159 is directly targeted to the Toc-complex in a homotypic interaction with atToc33 mediated by the G-domains of the two proteins (Hiltbrunner et al, 2001b;Bauer et al, 2002;Smith et al, 2002;Lee et al, 2003;Weibel et al, 2003). These results are supported by the crystal structure of the pea Toc34 homodimer identifying sequence motifs conserved in both atToc33 and atToc159 which are required for dimerization and chloroplast targeting of soluble atToc159 (Sun et al, 2002;Weibel et al, 2003).…”