Photoactivatable GFP tagging cassettes for protein-tracking studies in the budding yeast Saccharomyces cerevisiae.
Yeast cell biologists use a variety of fluorescent protein tags for determining protein localization and for measuring protein dynamics using fluorescence recovery after photobleaching (FRAP). Although many modern fluorescent proteins, such as those with photoactivatable and photoconvertible characteristics, have been developed, none has been exploited for studies in budding yeast. We describe here the construction of yeast-tagging vectors containing photoactivatable green fluorescent protein (PA-GFP) for analysis of protein behaviour. We tagged two yeast proteins, Erg6p and Num1p, with PA-GFP and demonstrated specific photoactivation of the fusion proteins in live cells. Fluorescence intensity measurements showed that a short 5 s exposure to 413 nm light is sufficient to produce the maximum level of activated GFP fluorescence. Local photoactivation of cortical Num1p-PA-GFP showed movement of the marked proteins, providing new insights into the behaviour of Num1p at the cell cortex. Since photoactivation can be achieved using standard mercury arc illumination, the PA-GFP tag represents a convenient and economical way to determine protein dynamics in the cell. Thus, the tagging modules should facilitate protein-tracking studies in a wide variety of cell biological processes in yeast.
Vorvis
Christina
C
Biology Department, University of Massachusetts at Amherst, 221 Morrill South, 611 North Pleasant Street, Amherst, MA 01003, USA.
Markus
Steven M
SM
Lee
Wei-Lih
WL
D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008780:Methyltransferases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D004271:DNA, Fungal; C071961:NUM1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase