Abstract: This chapter describes methods for studying different aspects of vacuole biogenesis and function. It presents three different approaches for visualizing the yeast vacuole: staining with vital dyes, using green fluorescent protein (GFP)-tagged vacuolar marker proteins, and immunofluorescence microscopy of formaldehyde-fixed cells. Because protein targeting to the vacuole is central to its function, the chapter presents various methods for assessing the fidelity of protein sorting using soluble and membrane-bound hydrolases, such as marker proteins. The chapter also discusses various techniques for determining whether an uncharacterized protein is associated with and/or degraded in the vacuole. The use of stage-specific transport mutants to identify the route by which a particular protein reaches the vacuole is also examined in the chapter. The chapter concludes with a description of methods for purifying vacuoles based on their low buoyant density to carry out biochemical studies, such as the assembly and activity of the V-ATPase.
Publication Year: 2002
Publication Date: 2002-01-01
Language: en
Type: review
Indexed In: ['crossref', 'pubmed']
Access and Citation
Cited By Count: 52
AI Researcher Chatbot
Get quick answers to your questions about the article from our AI researcher chatbot