Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Autor(es)

Gomes, Fernando ^[1] ; Turano, Helena ^[1] ; Ramos, Angelica ^[1] ; Netto, Luis E. S. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; n. 173 JUL 2021.
• Citações Web of Science: 0

Resumo

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards. (AU)

• Processo FAPESP: 17/09443-3 - Importação de peroxirredoxinas para distintos compartimentos mitocondriais: impactos em fisiologia e patologia.
• Beneficiário: Fernando Gomes
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 13/07937-8 - Redoxoma
• Beneficiário: Ohara Augusto
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 17/23839-7 - Investigação da ação bactericida da piocina S8: novas perspectivas para o tratamento de infecções causadas por cepas de Pseudomonas aeruginosa multirresistentes
• Beneficiário: Helena Gabriela Turano Gomes
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado