In vivo characterization of diatom multipartite plastid targeting signals

Abstract

Plastids of diatoms and related algae are delineated by four membranes: the outermost membrane (CER) is continuous with the endoplasmic reticulum while the inner two membranes are homologous to plastid envelope membranes of vascular plants and green algae. Proteins are transported into these plastids by pre-sequences that have two recognizable domains. To characterize targeting of polypeptides within diatom cells, we generated constructs encoding green fluorecent protein (GFP) fused to leader sequences. A fusion of GFP to the pre-sequence of BiP [an endoplasmic reticulum (ER)-localized chaperone]resulted in accumulation of GFP within the ER; a construct encoding the pre-sequence of a plastid protein fused to GFP was directed into the plastids. Additional constructs demonstrated that the N-terminal region of the bipartite plastid targeting pre-sequence was necessary for transport of polypeptides to the lumen of the ER, while the C-terminal region was shown to enable the proteins to traverse the plastid double envelope membrane. Our data strongly support the hypothesis of a multi-step plastid targeting process in chromophytic algae and raises questions about the continuity of the ER and CER and the function of the latter in polypeptide trafficking.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…Given the closely juxtaposed organelles, four plastid membranes and confined cytosolic spaces of P. tricornutum (Apt et al, 2002;Prihoda et al, 2012), an association between NR, plasma membrane transporters, and vacuolar membrane transporters may form as the proteins coalesce under specific conditions to facilitate the flux of NO 3 2 from the environment into and out of the vacuole. It remains to be determined whether free NO 3 2 is ever resident in the cytosol and whether, upon transport into the cell, it is either immediately reduced and transported into the chloroplasts or pumped into the vacuole.…”

Protein targeting into secondary plastids of chlorarachniophytes

“…This result confirms that the bipartite targeting peptide of BnAtpD, BnFdx1, and BnRpL28 are sufficient for targeting these preproteins into the plastids. The chlorarachniophytes use a bipartite targeting signal system that is similarly used in other secondary plastid-bearing eukaryotic groups, including heterokonts, cryptophytes, and apicomplexans (17,20,33). It is interesting to note that bipartite targeting peptides are commonly used for targeting proteins into the secondary plastids of diverse groups, even though the origins of their plastids are different.…”

“…Unlike the chlorarachniophyte plastids, the outermost plastid membrane is continuous with the ER in the plastids of heterokonts, cryptophytes, and haptophytes, and plastid preproteins are believed to be transported from the ER into the plastids via the membrane connection (9,35). It has been demonstrated in the heterokonts that the TPL functional domain localizes in the N-terminal portion (33). In euglenophytes and dinoflagellates, plastids are surrounded only by 3 membranes, and plastid preproteins are known to be transported from the ER to the plastids via Golgi bodies (8).…”

The glucans extracted with warm water from diatoms are mainly derived from intracellular chrysolaminaran and not extracellular polysaccharides

“…Immunolocalization P. tricornutum and T. pseudonana cells were fixed by freeze substitution and the polymerized resin blocks were sectioned using the protocols described by Apt et al (2002). The blocks of C. fusiformis cells prepared by freeze substitution were a kind gift of Drs Nicole Poulsen and Nils Kro¨ger of the University of Regensburg, Germany.…”