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Biophys J, December 2002, p. 3177-3187, Vol. 83, No. 6
and
*Laboratory for Fluorescence Dynamics, Department of
Biochemistry, University of Illinois in Urbana-Champaign,
Urbana, Illinois 61801 USA
Adenylate kinase (AK) is a ubiquitous enzyme that
regulates the homeostasis of adenine nucleotides in the cell. AK1
(long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its
N-terminus. It is hypothesized that these residues serve as a signal
for protein lipid modification and targeting of the protein to the
plasma membrane. To better understand the cellular function of these AK
isoforms, we have used several modern fluorescence techniques to
characterize these two isoforms of AK enzyme. We fused cytosolic
adenylate kinase (AK1) and its isoform (AK1) with enhanced green
fluorescence protein (EGFP) and expressed the chimera proteins in HeLa
cells. Using two-photon excitation scanning fluorescence imaging, we
were able to directly visualize the localization of AK1-EGFP and
AK1-EGFP in live cells. AK1-EGFP mainly localized on the plasma
membrane, whereas AK1-EGFP distributed throughout the cell except for
trace amounts in the nuclear membrane and some vesicles. We performed
fluorescence correlation spectroscopy measurements and photon-counting
histogram analysis in specific domains of live cells. For AK1-EGFP, we
observed only one diffusion component in the cytoplasm. For
AK1-EGFP, we observed two distinct diffusion components on the
plasma membrane. One corresponded to the free diffusing protein,
whereas the other represented the membrane-bound AK1-EGFP. The
diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect
to that of EGFP, which was 50% more than what we would expect for a
free diffusing AK1-EGFP. To rule out the possibility of oligomer
formation, we performed photon-counting histogram analysis to direct
analyze the brightness difference between AK1-EGFP and EGFP. From our
analysis, we concluded that cytoplasmic AK1-EGFP is monomeric.
fluorescence correlation spectroscopy proved to be a powerful technique
for quantitatively studying the mobility of the target protein in live
cells. This technology offers advantages in studying protein
interactions and function in the cell.
Biophys J, December 2002, p. 3177-3187, Vol. 83, No. 6
© 2002 by the Biophysical Society 0006-3495/02/12/3177/11 $2.00
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