Characterization of Chloroplast Protein Import without Tic56, a Component of the 1-Megadalton Translocon at the Inner Envelope Membrane of Chloroplasts

Abstract

We report on the characterization of Tic56, a unique component of the recently identified 1-MD translocon at the inner envelope membrane of chloroplasts (TIC) in Arabidopsis (Arabidopsis thaliana) comprising Tic20, Tic100, and Tic214. We isolated Tic56 by copurification with Tandem Affinity Purification-tagged Toc159 in the absence of precursor protein, indicating spontaneous and translocation-independent formation of the translocon at the outer envelope membrane of chloroplasts (TOC) and TIC supercomplexes. Tic56 mutant plants have an albino phenotype and are unable to grow without an external carbon source. Using specific enrichment of protein amino termini, we analyzed the tic56-1 and plastid protein import2 (toc159) mutants to assess the in vivo import capacity of plastids in mutants of an outer and inner envelope component of the anticipated TOC-TIC supercomplex. In both mutants, we observed processing of several import substrates belonging to various pathways. Our results suggest that despite the severe developmental defects, protein import into Tic56-deficient plastids is functional to a considerable degree, indicating the existence of alternative translocases at the inner envelope membrane.

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

“…This approach has already proven useful in the identification of TIC56 in a TOC159-containing super complex (17). Here, we identify KOC1 as a new component in this complex and show that it is required for full import activity and successful de-etiolation.…”

“…The 1MD complex associates with the translocating preprotein and forms a preprotein sensitive channel when reconstituted into planar lipid bilayers. TIC components (both absent and present in the 1 MD complex) co-purified with TOC159 suggesting that they cooperate in preprotein import (17).…”

“…The ClpC and cpHsp70 chaperones have both been reported to provide the driving force for preprotein import into the chloroplast (13)(14)(15). Recently, a 1MD complex has been described: in addition to TIC20 it includes TIC56, TIC100 as well as the chloroplast encoded TIC214 (YCF1) (16,17). The 1MD complex associates with the translocating preprotein and forms a preprotein sensitive channel when reconstituted into planar lipid bilayers.…”

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase

“…the development of the thylakoids). For example, mutations in TOC159 and TIC56 (Köhler et al, 2015) result in chlorotic seedlings and plastids with reduced and disorganized internal membranes. Mutations in components of a secondary destination translocase (e.g.…”

YCF1: A Green TIC: Response to the de Vries et al. Commentary

“…This system includes the Tic20 paralog Tic20-IV in Arabidopsis but lacks Tic56, Tic100, and Tic214 (Hirabayashi et al, 2011;Kikuchi et al, 2013;Parker et al, 2014;Nakai, 2015). Experimental data shown in recent work by Kö hler et al (2015) provide support for this model of more than one TIC complex. They found that the major TIC complex containing Tic56 shows very strong physical interaction with the major TOC complex containing the Toc159 receptor.…”