Clathrin in Chara australis: Molecular Analysis and Involvement in Charasome Degradation and Constitutive Endocytosis

Abstract

Charasomes are convoluted plasma membrane domains in characean green algae. They are known to form in response to light via secretion of trans-Golgi network (TGN) vesicles and local inhibition of endocytosis. Charasomes are involved in the acidification of their aqueous environment, thereby facilitating photosynthesis-dependent carbon uptake. Charasome formation is reversible to allow cells to adapt to different light conditions. Here, we show that darkness-induced degradation of charasomes involves the formation of coated pits and coated vesicles. The darkness-induced degradation of charasomes can be inhibited by 1–2 μM ikarugamycin (IKA), which is considered to be a specific inhibitor of clathrin-dependent endocytosis. At a much higher concentration (100 μM), IKA also significantly reduces the internalization of styryl dyes, indicating uptake via clathrin-coated vesicles (CV). We are the first to present evidence, based on fine structure investigation, that IKA does not interfere with the formation of clathrin coat, but inhibits the detachment and/or further processing of coated vesicles. Both charasome degradation and constitutive endocytosis are also significantly inhibited by sterol complexing agents (methyl-ß-cyclodextrin and filipin). The absence of an additive effect, when applied together with IKA, suggests that charasome degradation and constitutive endocytosis (measured via styryl dye uptake) is not inhibited due to membrane retrieval via lipid rafts, but due to clathrin coat formation requirement of a specific set of sterols. Analysis of Chara australis clathrin proteins revealed two heavy chains and several light chains with sequence peculiarities, suggesting functional and/or species specific differences. The data obtained indicate that clathrin plays a central role not only in constitutive endocytosis but also in the degradation of charasomes, thereby representing a valuable system for studying targeted exo- and endocytosis.

Dissecting the subcellular membrane proteome reveals enrichment of H+ (co-)transporters and vesicle trafficking proteins in acidic zones of Chara internodal cells

“…The significant enrichment of clathrin heavy chains and clathrin adaptor proteins at the acidic regions is consistent with the important role of these proteins in the formation as well as in the degradation of charasomes [ 22 , 65 ]. The formation of charasomes is light-dependent and under natural and culture conditions charasomes will be formed or degraded slowly, via a period of at least several days, and according to differences in light exposure due to (local) shading from the upper parts of the thallus.…”

“…RNA quality and integrity were checked using a spectrophotometer (NanoDrop, Thermo Scientific) as well as agarose gel electrophoresis. Pooled RNA extractions were send to BGI Genomics (Hongkong, China) for cDNA generation and de novo sequencing of the Chara australis transcriptome (single replicate) using the Illumina Hiseq 2000 technology [ 22 , 35 ]. Raw sequence data were filtered and the resulting clean reads were assembled to unigenes using Trinity software [ 36 ]).…”

“…Charasomes are degraded upon dark incubation and they re-appear when cells are exposed to light [ 22 – 24 ]. Recently it was shown that charasomes are formed via exocytosis of trans Golgi network-derived material and local inhibition of endocytosis (coated vesicle detachment) that probably involves a phosphoinositide 4 kinase [ 25 ], whereas degradation of charasomes occurs via clathrin-dependent membrane retrieval [ 22 ].…”

Transcriptome Analysis Reveals Candidate Genes Associated with Leaf Etiolation of a Cytoplasmic Male Sterility Line in Chinese Cabbage (Brassica Rapa L. ssp. Pekinensis)

“…Thus, the dysfunction of chloroplasts usually results in the loss of leaf green color [ 16 ]. Leaf etiolation of mutants is determined by the expression level of the key genes involved in chloroplast development [ 17 ]. Several genes affecting chloroplast development have been reported, and their role in leaf etiolation has been identified through studies of leaf mutants in Arabidopsis thaliana [ 18 ], rice [ 19 ], and tomato [ 20 ].…”

Effect of Carbon Nanotube-Metal Hybrid Particle Exposure to Freshwater Algae Chlamydomonas reinhardtii

“…This could account for the increased toxicity of the CNT-Ag contrasted with the other CNT variants. The uptake mechanism of algae for carbon particles is not well understood, although clathrin has been isolated from algae and this protein is a constituent of basic cellular internalization 59 , and simple endocytosis is believed to be an evolution ancient trait in plants 60 . This fact, combined with the greater internalization of CNT-Ag most likely accounts for the toxicity, although the differential mechanism of uptake still needs to be determined.…”