Proteins labeling within cells

Recently, Beatty and Tirrell [201] relied on the simultaneous or sequential addition of two reactive Met analogs, Aha and Hpg, to enable the fluorescent tagging of two protein populations within cells. The first demonstration of two-dye labeling of metabolically tagged cells was described in 2007 by Chang and co-workers [202], who used flow cytometry to show that cells treated with two reactive sugars could be labeled with distinct fluorophores. 

Advances in pulsed lasers, microscopy and computer imaging, and the development of labelling techniques in which the donor and acceptor fluorophores become part of the biomolecules themselves have enabled the visualisation of dynamic protein interactions within living cells. 

In discussing photoaffinity labeling experiments with radioactive reagents, I shall focus mainly on the labeling of protein receptors with small photola-bile ligands. The same principles may be applied to related experiments. The general approach to identifying a receptor in a mixture of proteins is first described. The mixture may be extremely complex, e.g. whole cells or a membrane preparation, or relatively simple, e.g. a purified multisubunit protein. Second, experiments to locate a label within a protein are described. This may be at the level of a low resolution peptide map or in favorable cases the identification of a labeled amino acid residue. 

Although autoradiography Is rarely used today to localize proteins within cells, these early experiments Illustrate the two basic requirements for any assay of intercompartmental transport. First, it is necessary to label a cohort of proteins In an early compartment so that their subsequent transfer to later compartments can be followed with time. Second, It Is necessary to have a way to Identify the compartment In which a labeled protein resides. Here we describe two modern experimental procedures for observing the Intracellular trafficking of a secretory protein In almost any type of cell. 

Our kinetic studies of mutant PrPs synthesized in CHO cells suggest that individual steps in formation of PrP may take place in at least two different cellular locations (Fig. 5). Because mutant PrPs become PIPLC-resistant within minutes of synthesis in pulse-labeling experiments, this early step must take place in the ER. Consistent with this conclusion, acquisition of PIPLC resistance is not affected by treatment of cells with brefeldin A or by incubation at 18°C, manipulations that block exit of proteins beyond the Golgi (Daude et al, 1997). In contrast, detergent insolubility and protease resistance, which do not develop until later times of chase, and are reduced by brefeldin A and 18°C incubation, are likely to be acquired after arrival of the protein at the cell surface, either on the plasma membrane itself or in endocytic compartments. Raft domains may be involved in these changes (unpublished data). 

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