Isolation of plastids and mitochondria from Chromera velia

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F19%3A00520514" target="_blank" >RIV/60077344:_____/19:00520514 - isvavai.cz</a>

Alternative codes found

RIV/60076658:12310/19:43899743

Result on the web

<a href="https://link.springer.com/content/pdf/10.1007/s00425-019-03259-3.pdf" target="_blank" >https://link.springer.com/content/pdf/10.1007/s00425-019-03259-3.pdf</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s00425-019-03259-3" target="_blank" >10.1007/s00425-019-03259-3</a>

Result language

angličtina

Original language name

Isolation of plastids and mitochondria from Chromera velia

Original language description

Main conclusionWe present an easy and effective procedure to purify plastids and mitochondria from Chromera velia. Our method enables downstream analyses of protein and metabolite content of the organelles.AbstractChromerids are alveolate algae that are the closest known phototrophic relatives to apicomplexan parasites such as Plasmodium or Toxoplasma. While genomic and transcriptomic resources for chromerids are in place, tools and experimental conditions for proteomic studies have not been developed yet. Here we describe a rapid and efficient protocol for simultaneous isolation of plastids and mitochondria from the chromerid alga Chromera velia. This procedure involves enzymatic treatment and breakage of cells, followed by differential centrifugation. While plastids sediment in the first centrifugation step, mitochondria remain in the supernatant. Subsequently, plastids can be purified from the crude pellet by centrifugation on a discontinuous 60%/70% sucrose density gradient, while mitochondria can be obtained by centrifugation on a discontinuous 33%/80% Percoll density gradient. Isolated plastids are autofluorescent, and their multi-membrane structure was confirmed by transmission electron microscopy. Fluorescent optical microscopy was used to identify isolated mitochondria stained with MitoTracker(TM) green, while their intactness and membrane potential were confirmed by staining with MitoTracker(TM) orange CMTMRos. Total proteins were extracted from isolated organellar fractions, and the purity of isolated organelles was confirmed using immunoblotting. Antibodies against the beta subunit of the mitochondrial ATP synthase and the plastid protochlorophyllide oxidoreductase did not cross-react on immunoblots, suggesting that each organellar fraction is free of the residues of the other. The presented protocol represents an essential step for further proteomic, organellar, and cell biological studies of C. velia and can be employed, with minor optimizations, in other thick-walled unicellular algae.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

10611 - Plant sciences, botany

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year

2019

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Planta

ISSN

0032-0935

e-ISSN

—

Volume of the periodical

250

Issue of the periodical within the volume

5

Country of publishing house

DE - GERMANY

Number of pages

11

Pages from-to

1731-1741

UT code for WoS article

000491965700024

EID of the result in the Scopus database

2-s2.0-85070799974