In Silico and In Vivo Investigations of Proteins of a Minimized Eukaryotic Cytoplasm

Abstract

Algae with secondary plastids such as diatoms maintain two different eukaryotic cytoplasms. One of them, the so-called periplastidal compartment (PPC), is the naturally minimized cytoplasm of a eukaryotic endosymbiont. In order to investigate the protein composition of the PPC of diatoms, we applied knowledge of the targeting signals of PPC-directed proteins in searches of the genome data for proteins acting in the PPC and proved their in vivo localization via expressing green fluorescent protein (GFP) fusions. Our investigation increased the knowledge of the protein content of the PPC approximately 3-fold and thereby indicated that this narrow compartment was functionally reduced to some important cellular functions with nearly no housekeeping biochemical pathways.

Evidence for glycoprotein transport into complex plastids

“…GFP fusion proteins of all constructs accumulate in the classical blob-like structure (Fig. 2C), which is typical for a localization within the periplastidal compartment (9,12,28,29). Altogether, these results demonstrate that even very bulky, highly N-glycosylated proteins can be transported across the second plastid membrane into the PPC.…”

“…Besides such synthetic constructs, we screened all known endogenous PPC proteins (9,10,13,16,(29)(30)(31) for potential N-glycosylation sites using the program NetNGlyc 1.0. Interestingly, 23% of the 52 proteins possess potential N-glycosylation sites with high confidence predictions (++) within the mature, soluble protein (Table S1).…”

“…S1). The protein sPub (symbiont-specific PNGase/UBA or UBX) is a potential adapter protein of the AAA-ATPase Cdc48 containing an additional thioredoxin domain and was previously shown to be localized within the PPC (29). The protein sGt8 (symbiont-specific glycosyltransferase family 8) shows homology to glycosyltransferases of the GT8 family.…”

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

“…1). However, only a handful of PPC-targeted proteins are known (for example, see refs [18][19][20][21] and the true extent of this dependence is unclear. Indeed, why nucleomorph genomes have been retained at all is a long-standing mystery of plastid evolution.…”

“…On balance, the PPC of cryptophytes and chlorarachniophytes is highly reduced, but has retained an unexpectedly broad range of biochemical processes. These data provide the basis for addressing many fundamental questions about algal cell biology, including how many homologues of G. theta and B. natans PPC proteins are retained in the nucleomorphlacking PPC of algae such as diatoms and haptophytes 21 , and what exactly are the biochemical determinants of the protein trafficking pathways in cryptophytes, chlorarachniophytes and other secondary plastid-bearing algae 1,27 . Making sense of the hundreds of predicted PPC and nucleomorph proteins in G. theta and B. natans with unknown functions (Supplementary Table 1.9.4.1.1) will be a substantial challenge.…”