A nucleus‐encoded maize protein with sigma factor activity accumulates in mitochondria and chloroplasts

Abstract

SummaryPlants contain nuclear gene families that encode proteins related to the principal sigma factors of eubacteria. As sigma factors function in transcription, the plant proteins have been presumed or demonstrated to associate with the eubacteria-like RNA polymerase of chloroplasts. In maize, ®ve sig cDNA sequences have been reported, and four of the products are present in plastids as predicted. However, in vitro chloroplast import assays and computer algorithms gave ambiguous results with the ®fth protein, ZmSig2B. Unlike the other maize sigma factors, ZmSig2B is expressed throughout developing seedling leaves, as well as in roots and etiolated tissues. To determine the subcellular location of ZmSig2B, we have now used immunoblot assays to show that it co-puri®es with both mitochondria and plastids. Its NH 2 -terminal 153 amino acids, translationally fused to green¯uorescent protein (GFP), targeted GFP to chloroplasts and mitochondria in bombarded maize leaves. A putative role for ZmSig2B in mitochondrial transcription is supported by its presence in a maize mitochondrial transcription extract. ZmSig2B also exhibits the expected properties of a chloroplast sigma factor: recombinant ZmSig2B binds to a chloroplast promoter and initiates transcription in vitro when combined with Escherichia coli core RNA polymerase. Therefore ZmSig2B is an unusual nucleus-encoded sigma factor that appears to function in both chloroplasts and mitochondria.

AtSig5 Is an Essential Nucleus-Encoded Arabidopsis σ-Like Factor

“…Because the amino acid sequence of AtSig5 bears a strong resemblance to that of eubacterial -factors, it had been proposed (but not experimentally demonstrated) that AtSig5 is a chloroplastlocalized protein that assembles with the eubacteriallike PEP RNA polymerase in plastids. However, the recent unexpected discovery of a maize -factor targeted to mitochondria as well as to plastids has emphasized how important it is to generate experimental evidence for plant -factor subcellular localization (Beardslee et al, 2002). We used GFP fusion proteins to demonstrate that, in transient expression assays in rosette leaves, an AtSig5 protein initiated at the upstream in-frame M1 Met could be targeted to both mitochondria and plastids, whereas the shorter protein initiated at M2 was exclusively plastid targeted.…”

“…As controls to demonstrate chloroplast and mitochondrial targeting patterns, GFP was fused to the chloroplast transit peptide sequence of Rubisco small subunit or to the mitochondrial targeting sequence of the F 0 F 1 ATPase ␥-subunit (Chang et al, 1999;Beardslee et al, 2002). Strong GFP fluorescence (Fig.…”

“…Consistent with this model, many plant -factors contain transit peptides capable of targeting the proteins into chloroplasts (Isono et al, 1997;Kanamaru et al, 1999;Lahiri et al, 1999;Fujiwara et al, 2000;Lahiri and Allison, 2000). Interestingly, the maize -factor, ZmSig2B, accumulates not only in chloroplasts but also in mitochondria, and contains an NH 2 -terminal sequence able to target fused green fluorescent protein (GFP) into mitochondria in transient expression assays (Beardslee et al, 2002). A specific role for this -factor in mitochondria remains to be elucidated.…”

“…Genetic analyses of the eubacterial -factors, as well as more recent structural studies, have supported the importance of these conserved domains in such eubacterial transcription functions as promoter recognition, interaction with the RNA polymerase core enzyme, and transcription initiation (Gross et al, 1998;Burgess and Anthony, 2001;Campbell et al, 2002;Murakami et al, 2002). The conservation of primary structure among plant and eubacterial -factors is sufficiently high to permit recombinant plant -factors to function with E. coli core RNA polymerase in vitro (Kestermann et al, 1998;Hakimi et al, 2000;Beardslee et al, 2002). Therefore, it is assumed (although not yet experimentally demonstrated) that these nucleus-encoded plant -factors assemble in plastids with the PEP RNA polymerase to effect promoter recognition and transcription initiation.…”

Plant organellar protein targeting: a traffic plan still under construction

“…Likewise, cellular and developmental factors can influence the activity of one translation initiation site over another. Some reports have described dual targeting in a GFP reporter assay as a non-simultaneous process, with predominant localization to mitochondria observed in one experiment, and to plastids when the experiment is repeated [32]. These sorts of experimental ambiguities are not uncommon and can reflect genuine fluctuations in the system.…”