Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching.
نویسندگان
چکیده
Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane-localized fluorophore with the cytosol during fluorescence recovery after photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma membrane-localized wild-type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 microm x 1 microm bleach region-of-interest (ROI) and a 0.5 microm x 0.5 microm bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in the yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane-associated fluorophores using FRAP on commercial confocal laser scanning microscopes.
منابع مشابه
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 ...
متن کاملMembrane bound protein diffusion viewed by fluorescence recovery after bleaching experiments : models analysis
Membrane bound protein diffusion viewed by fluorescence recovery after bleaching experiments : models analysis. Abstract Diffusion processes in biological membranes are of interest to understand the macromolecular organisation and function of several molecules. Fluorescence Recovery After Photobleaching (FRAP) has been widely used as a method to analyse this processes using classical Brownian d...
متن کاملCharacterization of Yeast Protein Enzymatic Hydrolysis and Autolysis in Saccharomyces cerevisiae and Kluyveromyces marxianus
Protein recovery under sonication treatment and autolysis, also protein hydrolysis progress during enzymatic hydrolysis (using trypsin and chymotrypsin) and autolysis (using endogenous enzymes) were investigated in Saccharomyces cerevisiae and Kluyveromyces marxianus. Crude protein content of dried yeast cells were 53.22% and 45.6% for S.cerevisiae and K.marxianus, respectively. After 96 hrs of...
متن کاملDetermination of diffusion coefficients in live cells using fluorescence recovery after photobleaching with wide-field fluorescence microscopy
Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system a...
متن کاملStatistical Optimization of Conditions for Maximize Production of Mannan by Saccharomyces Cerevisiae Using Response Surface Methodology
In view of the increase in Saccharomyces cerevisiae mannan content, the culture condition for S.cerevisiae were optimized in this study. The influence of culture condition such as original pH, inoculum size, and temperature on mannan production were evaluated using Response surface methodology. The mathematical model was established by the quadratic rotary combination design. with the order of ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Physical biology
دوره 7 2 شماره
صفحات -
تاریخ انتشار 2010