Bj 2002 Graton Mantulin Glaser

Cellular Characterization of Adenylate Kinase and Its Isoform: Two-Photon Excitation Fluorescence Imaging and Fluorescence Correlation Spectroscopy Qiaoqiao Ruan,* † Yan Chen,* Enrico Gratton,* Michael Glaser, † and William W. Mantulin* † *Laboratory for Fluorescence Dynamics, Department of † Biochemistry, University of Illinois in Urbana-Champaign, Urbana, Illinois 61801 USA ABST RACT Ade nyla te kina se (AK) is a ubiq uitous enz yme tha t regu lates the homeost asis of ade ninenucleotidesin 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 enh anc ed green fluo res cen ce prot ein (EGFP) and expr esse d the chimera prot eins in HeL a cell s. Usin g two- phot on exc itat ion sca nnin g fluorescence imag ing, we wer e able to dire ctly visuali ze the loca lization of AK1- EGF P 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 dif fus ion co mpo ne nt in the cyt opl as m. For AK1 -EGFP, we obs erved two dis tin ct diffusion co mpo ne nts on the pla smamembran e. 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- EGF P is monomeri c. fluo rescence cor rel atio n spec tros copy pr oved to be a powe rful te chn ique fo r quant itat ivel y studyi ng the mobi lity of the targ et prot ein in live cel ls. This techn olog y offers advan tage s in stud ying prot ein inter acti ons and func tion in the cell . INTRODUCTION Adenylate kinase (AK) is a ubiquitous monomeric enzyme that catalyzes the following reaction: Mg 2 -ATP AMP 7 Mg 2 -ADP ADP (Noda, 1973). The cell uses this react ion to conve rt AMP to ADP (Glaser et al ., 1975) , ther eby regulat ing aden ine nucl eotide levels. AK is als o invo lved in other reac tions, such as the biosynthes is of phospholipids (Goelz and Cronan, 1982). In vertebrates, thr ee isoz ymes of AK have been char acte riz ed: AK1 is cytoplasmic, AK2 is localized in the intermembrane space of mitochondria, and AK3 is localized in the mitochondrial matrix. Recently, a long form of AK1 protein from murine cells, AK1, was identified. Its amino acid sequence was identical to that of the murine cytoplasmic AK1, except for the addi ti on of 18 ami no aci ds (MGCCVSSEPQEE- GGRKTG) at the N-terminus . The transcr iption level of the AK1 gene was found to be upregulated by p53, whereas that of the AK1 gene was not. This observation suggested that AK1 might have some nove l biologi cal func tions other than its conventional function of regulating nucleotide levels. For example, it may be involved in the cell cycle arrest process (Collavin et al., 1999). We are interested in the effect of the additional 18 amino acid residues on the biological function of AK1. It was hypothesized that the 18 additional residues might serve as a signal for protein lipid modific ation and targeting of the protein to the plasma membran e, based on the N-myristoylation consensus motif. Protein N-myristoylation is the result of the co-translational addition of myristic acid to a Gly residue at the extreme N-terminus after removal of the initiating Met (Utsumi et al., 2001). In general, to direct protein N-myristoylation, the N-terminal consensus motifs such as Met-Gly-X-X-X Ser- X-X-X (Johnson et al., 1994) or Met-Gly-X-X-X Thr-X- X-X (Boutin, 1997) are preferred. Fluorescence correlation spectroscopy (FCS) was first intro- duced by Webb and his coworkers in 1972 (Magde et al., 1972). It has evolved as a powerful method to study particle dynamics on a single-molecule level in part because of recent technological advances (such as confocal microscopy, multi- photo n laser excita tion ). FCS uses the autocor relat ion functi on to characterize fluorescence intensity fluctuations in the observation volume and it can be used to study particle diffusion (Fahey et al., 1977; Koppel et al., 1976), chemical kinetics (Haupts et al., 1998; Starr and Thompson, 2001), and molec- ular aggregation (Palmer and Thompson, 1987; Qian and El- son, 1990). In recen t years, several labor atories have intro- duced FCS studies on live cells. Fluorescently labeled particles were first loaded into cells or labeled on the surface of the cells; the dynamics of the fluorescent dyes in the cells were then studied with FCS. Successful application of FCS in the cell provides a new experimental approach to quantitatively study the diffusion rates of cellular mole- Submitted May 23, 2002, and accepted for publication July 24, 2002. Address reprint requests to Dr. William W. Mantulin, 1110 W. Green St., Urbana, IL 61801-3080. Tel.: 217-244-5620; Fax: 217-244-7187; E-mail: [email protected]. © 2002 by the Biophysic al Societ y 0006-3 495/0 2/12/ 3177/ 11 $2.00 3177 Bioph ysica l Journ al Volume 83 Dece mbe r 2002 3177 –318 7

Bj 2002 Graton Mantulin Glaser

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