Journal
JOURNAL OF CELL SCIENCE
Volume 117, Issue 14, Pages 3049-3059Publisher
COMPANY BIOLOGISTS LTD
DOI: 10.1242/jcs.01167
Keywords
protein movement; arrestin; Xenopus; transgenesis
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Funding
- NEI NIH HHS [EY02660, EY12910, R01 EY016453, EY12975] Funding Source: Medline
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The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which similar to40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.
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