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.

FIG. 1.—

The PPC and cell structure/plastid compartmentalization of the diatom P. tricornutum. (A) Postulated functions of PPC-localized proteins are indicated in black boxes. s, symbiontic (PPC-localized); sDer1-1/1-2, degradation at the ER; sUfd1, ubiquitin fusion degradation; sUba1, ubiquitin activating E1; sUbc, ubiquitin conjugating E2; ptE3P, P. tricornutum ubiquitin ligase E3 of the PPC; Ub, ubiquitin; ptDUP, P. tricornutum deubiquitinating enzyme of the PPC; sCdc48-1/2, cell division cycle protein; sPUB, PUB and thioredoxin domain containing protein; sHsp70, heat shock protein; sDTC, DnaJ and TPR domain-containing protein; sDPC, DnaJ and PDI domain-containing protein; sSec14, putative lipid transfer protein sTrxH, thioredoxin; sNTRC, NADPH depending thioredoxin reductase containing N-terminal thioredoxin domain; sDrp, dynamin-related protein; sα7/sβ2/6/7, proteasomal 20S components of the alpha and beta type; sTLP-1, trypsin-like serine protease; sSMC, structural maintenance of the chromosome-like protein; sPRP, pentapeptide repeats containing protein; sPEL, pectin esterase domain-containing protein; sP4H, prolyl-4-hydroxylase; 6PGDH, 6-phosphogluconolactone dehydrogenase; sαCA-1/2, alpha carbonic anhydrase; sORF139/261/532a/534, open reading frame (Guillardia theta nucleomorph-encoded ORF homolog); ptOmp85, outer membrane protein. Superscript numbers indicate proteins localized in previous studies: 1, (Gould et al. 2006a); 2, (Sommer et al. 2007); 3, (Hempel et al. 2010); 4, (Gruber et al. 2009); 5, (Weber et al. 2009); 6, (Bullmann et al. 2010). (B) Phaeodactylum tricornutum possesses an aliform-shaped secondary plastid surrounded by four membranes. The outermost membrane is in continuum with the host rough endoplasmic reticulum (rER). The space between the two inner- and outermost membrane pairs (PPC) represents the former red algal cytoplasm of the endosymbiont. cERM, chloroplast ER membrane; cER, chloroplast ER; PPM, periplastidal membrane; PPC, periplastidal compartment; OEM, plastid outer envelope membrane; IMS, plastid intermembrane space; IEM, plastid inner envelope membrane; Pl, plastid; Nu, nucleus; Mi, mitochondrion; GA, Golgi apparatus.

FIG. 2.—

In vivo localization studies of P. tricornutum BTS/FL sequences fused to eGFP. Homologous overexpression of BTS- or full-length (FL)-GFP fusion proteins in all cases led to a characteristic 'blob-like' GFP-fluorescence pattern, known to correspond to a typical PPC-localization. The blob-like structure is due to a median constriction of the two innermost membranes of the plastid (OEM/IEM), which leads to a widening of the PPC. TL, transmitted light; Merge, overlay of plastid autofluorescence (red) and GFP fluorescence (green). The scale bar represents 10 μm. For further information about the proteins, see text and the legend of figure 1A.