Molecular Characterization of the PEND Protein, a Novel bZIP Protein Present in the Envelope Membrane That Is the Site of Nucleoid Replication in Developing Plastids

Abstract

Plastid nucleoids are known to bind to the envelope membrane in developing chloroplasts. Here, plastid DNA is extensively replicated. We previously detected a DNA binding protein in the inner envelope membranes of developing plastids in pea and named it PEND (for plastid envelope DNA binding) protein. In this study, we report on the structure and molecular characterization of a cDNA for the PEND protein. As a result of screening cDNA libraries in lambdagt11 with one of the target sequences of the PEND protein as a probe, we obtained a clone (PD2) for a novel DNA binding protein consisting of 633 amino acid residues. Analysis of the N-terminal sequence of the purified PEND protein indicated that the transit peptide is just 16 residues long. The PEND protein was detected specifically in the plastid envelope membrane of young unopened leaf buds by immunoblot analysis. The PEND protein consists of a basic region plus zipper region, an unprecedented sextuple repeat region, and a putative membrane-spanning region. The basic region with a zipper region seems to have diverged from that of other plant transcription factors. In addition, the PEND protein could be a distant homolog of the trans-Golgi network integral membrane proteins. The PEND protein is therefore a novel type of DNA binding protein that binds to the membrane as an intrinsic membrane protein.

Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

“…Interestingly, sulfite reductase was not identified from previous proteome investigations of chloroplast envelope membranes 9,39,40 despite biochemical evidence suggesting nucleoids are tightly associated with envelope membranes. 15,41,42 Another class of nucleoid-associated proteins identified in this investigation were DNA gyrase-topoisomerases. Like sulfite reductase, DNA gyrases were abundant in Triton-insoluble fractions based upon intensities of CBB stained bands 7, 8, and 12.…”

“…[12][13][14] The subplastidial location of nucleoids is a matter of speculation, although biochemical evidence indicates an association with stromal-side of the envelope membrane. 15 In addition to nucleiods, membrane-depleted chloroplast subfractions likely contain high molecular weight protein complexes since the multi-enzyme ACCase was also prevalent. To verify this and begin developing mechanistic models for multi-enyzme complexes and plastid nucleoids we performed the first proteomic characterization of a membrane-depleted, high-density chloroplast fraction.…”

New insights into plastid nucleoid structure and functionality

“…The plastid envelope DNA binding protein (PEND) is a 70 kDa membrane-spanning protein with a basic region plus leucine zipper (bZIP) domain that forms dimers in vivo which seem to tether the nucleoids to the plastid inner envelope (Sato et al , 1998. The PEND protein was initially discovered in developing pea chloroplasts .…”

The Core of Chloroplast Nucleoids Contains Architectural SWIB Domain Proteins

“…Number, position, and compactness of nucleoids are known to parallel the changes in gene expression occurring during chloroplast development. Two DNA binding proteins, the plastid envelope DNA binding protein (PEND) (Sato et al, 1998) and the matrix attachment region binding filamentlike protein1 (MFP1) (Jeong et al, 2003), are known to be involved in redistribution of the plastid nucleoids from the envelope membrane to the thylakoids during the transition from proplastids to chloroplasts. However, proteins involved in regulation of nucleoid morphology and compactness and corresponding to the bacterial HU (for histone-like protein from Escherichia coli strain U93) and HU-like proteins have not been identified hitherto (Sato et al, 2003;Sakai et al, 2004).…”