Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae

Abstract

SUMMARYPlant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate-rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF-MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density-gradient ultracentrifugation. The vacuole-containing fraction was subjected to SDS-PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non-lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o-methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin-tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.

Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage

“…A compartment called the acidocalcisome has been described in C. reinhardtii (Ruiz et al, 2001). This compartment contains polyphosphate and metals and is a specialized vacuolar structure based on proteomic analysis of Cyanidioschyzon merolae acidocalcisomes that identified V-ATPase subunits (Yagisawa et al, 2009). In addition, at least two candidate metal transporters, including a protein related to S. cerevisiae Ccc1 and a cation diffusion facilitator family member (putative Zn transporter), were described.…”

Intracellular Structure of the Unicellular Red Alga <i>Cyanidioschyzon merolae</i> in Response to Phosphate Depletion and Resupplementation

“…We speculate that the contact is related to a vacuolar function in lipid degradation. The C. merolae lipases were identified in a previous vacuolar proteomics study (Yagisawa et al 2009), suggesting that vacuoles have a role in lipid degradation. In algae, lipid profiles change during phosphate starvation (Khozin-Goldberg and Co- Following phosphate resupplementation, electrondense granules/vesicles appeared in the cytoplasm.…”

“…C. merolae vacuoles likely contain acid phosphatases and a protein homologous to yeast endopolyphosphatase Ppn1 (Yagisawa et al 2009). We did not see electron-dense substances transferred to other part of the cells, which suggests that polyphosphate may be degraded in the vacuoles by some of these enzymes (Fig.…”

“…Our previous vacuolar proteomics study has suggested that C. merolae vacuoles harbor a protein partially similar to Vtc1 (Yagisawa et al 2009); however, Vtc4 with its conserved catalytic domain has not been found in the genome. Thus, where and how polyphosphate is synthesized in this alga remain unknown.…”

Genome Size of the Ultrasmall Unicellular Freshwater Green Alga, <i>Medakamo hakoo</i> 311, as Determined by Staining with 4′,6-Diamidino-2-phenylindole after Microwave Oven Treatments: II. Comparison with <i>Cyanidioschyzon merolae</i>, <i>Saccharomyces cerevisiae</i> (<i>n</i>, 2<i>n</i>), and <i>Chlorella variabilis</i>

“…Also, C. merolae has a minimum set of organelles: three types of double-membrane-bound organelles (the cell nucleus, mitochondrion, and plastid), and four types of single-membrane-bound organelles [endoplasmic reticulum (ER), Golgi apparatus, peroxisome (microbody), and lysosome]. These organelles are essential for fulfilling the functions of eukaryotic cells and all of them proliferate by division (Kuroiwa et al 1994, Yagisawa et al 2009, Imoto et al 2013. In addition, the cellular and organelle divisions of C. merolae can be completely synchronized by light and dark cycles (Terui et al1995, Imoto et al 2013.…”