Lysosome-related organelles (LROs) are a class of heterogeneous organelles conserved in eukaryotes that primarily play a role in storage and secretion. An important function of LROs is to mediate metal homeostasis. Chlamydomonas reinhardtii is a model organism for studying metal ion metabolism; however, structural and functional analyses of LROs in C. reinhardtii are insufficient. Here, we optimized a method for purifying these organelles from 2 populations of cells: stationary phase or overloaded with iron. The morphology, elemental content, and lysosomal activities differed between the 2 preparations, even though both have phosphorus and metal ion storage functions. LROs in stationary phase cells had multiple non-membrane-bound polyphosphate granules to store phosphorus. Those in iron-overloaded cells were similar to acidocalcisomes (ACs), which have a boundary membrane and contain 1 or 2 large polyphosphate granules to store more phosphorus. We established a method for quantifying the capacity of LROs to sequester individual trace metals. Based on a comparative proteomic analysis of these 2 types of LROs, we present a comprehensive AC proteome and identified 113 putative AC proteins. The methods and protein inventories provide a framework for studying the biogenesis and modification of LROs and the mechanisms by which they participate in regulating metal ion metabolism.

Cilia/flagella are conserved eukaryotic organelles that play an important role in the control of cell motility and detection of environmental cues. However, the molecular mechanisms underlying ciliary/flagellar assembly, maintenance, disassembly, and signal transduction are not yet completely understood. Recent studies demonstrated that post-translational modifications (PTMs) such as phosphorylation, methylation, glutamylation, and ubiquitination are involved in these processes. In this mini review, we present a summary of research progress in ciliary/flagellar protein ubiquitination, including the ubiquitin conjugation system identified by proteomics as well as the role of ciliary/flagellar protein ubiquitination in flagellar disassembly, motility, and signal transduction. Moreover, we described putative further research directions in the study of ciliary/flagellar protein ubiquitination.

The flagellar (ciliary) membrane of Chlamydomonas reinhardtii is continuous with the plasma membrane but maintains a distinct protein composition through the ciliary gate mechanism; however, a systematic comparison of the membrane proteomes of these two compartments has not been reported. Here, we performed comparative proteomics on highly enriched plasma and flagellar membrane fractions and identified 2779 proteins, with hundreds differentially abundant. Plasma membrane-enriched proteins were mainly involved in proton transport and ATP synthesis, whereas flagellar membrane-enriched proteins were specialized for cilium movement, axoneme assembly, and intraflagellar transport. Fluorescence localization validated the proteomic data and revealed previously uncharacterized subcellular patterns. Notably, PLD1 showed dual localization at the plasma membrane and punctate cytoplasmic structures, and FAP215 (validated by subflagellar fractionation) displayed a broader flagellar signal than the central microtubule protein FAP178, consistent with its localization in the flagellar matrix or membrane. This work provides the first systematic comparison of plasma and flagellar membrane proteomes in Chlamydomonas, offering a valuable resource for understanding flagellar membrane specialization.

Biosynthetic gas vesicles (GVs), as novel nanoscale ultrasound contrast agents, exhibit unique potential in biomedical ultrasound imaging. For example, they are expected to have better tissue penetration through the tumor vasculature for detecting tumor cells by the design of GV-based acoustic probes. Of all these GVs, GVs from Halobacterium sp. NRC-1 possess the largest size (over 200 nm) and are nearly spherical in shape, endowing them with stronger acoustic signals and better tumor penetration. However, their genetic manipulation is relatively difficult due to the requirement of a high-salt cytoplasmic environment for their expression and assembly, limiting the application of biosynthetic technology for modulating their structural features in heterologous host cells. In this study, we cloned the gene cluster encoding GVs from Halobacterium sp. NRC-1 and transformed it into Haloferax volcanii, an archaeal species naturally incapable of producing GVs. The genetically engineered Haloferax volcanii successfully synthesized functional GVs (GV_vol) with a similar size and shape to naturally synthesized GVs from Halobacterium sp. NRC-1 (GV_halo). The ultrasound imaging properties of GV_vol heterologously synthesized in Haloferax volcanii were compared with naturally synthesized GV_halo in vitro and in vivo, showing that GV_vol could achieve a mean signal intensity of 113.6 ± 0.9 a.u. in vitro and a peak intensity of 121.5 ± 0.8 a.u. in vivo in the kidney, compared with 115.7 ± 0.5 a.u. and 119.0 ± 0.5 a.u. for GV_halo, respectively. These findings confirm the functional integrity of heterologously synthesized GV_vol and its potential for biomedical applications. Our study provides a solid experimental foundation for genetically tailoring Halobacterium GV properties to optimize biomedical imaging performance.