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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Full text
Author(s):
Gomes, Fernando [1] ; Turano, Helena [1] ; Ramos, Angelica [1] ; Netto, Luis E. S. [1]
Total Authors: 4
Affiliation:
[1] Univ Sao Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; n. 173 JUL 2021.
Web of Science Citations: 0
Abstract

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)

FAPESP's process: 17/09443-3 - Importing of peroxiredoxins to distinct mitochondrial compartments: impacts on physiology and pathology
Grantee:Fernando Gomes
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
Grantee:Ohara Augusto
Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
FAPESP's process: 17/23839-7 - Investigation of the S8 pyocin bactericidal activity: new perspectives for the treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa strains
Grantee:Helena Gabriela Turano Gomes
Support Opportunities: Scholarships in Brazil - Post-Doctoral