Cyclic AMP controls several signalling cascades within cells, and changes in the amounts of this second messenger have an essential role in many cellular events. Here we describe a new methodology for monitoring the fluctuations of cAMP in living cells. By tagging the cAMP effector protein kinase A with two suitable green fluorescent protein mutants, we have generated a probe in which the fluorescence resonance energy transfer between the two fluorescent moieties is dependent on the levels of cAMP. This new methodology opens the way to the elucidation of the biochemistry of cAMP in vivo.

Abstract, A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca 2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca 2÷ buffering capacity of the intracellular Ca 2÷ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. One-and two-dimensional Western blots revealed the transfected to exceed the endogenous CR of ~3.5-fold and to maintain its Ca 2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca 2÷ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura-2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca2+-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca 2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca 2÷ buffering within the IP3-sensitive, rapidly exchanging, Ca 2÷ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca 2+ capacity of the stores is not the main limiting factor of individual IP3-mediated Ca 2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca 2+ buffering within the IP3-sensitive stores accounts for ~45 % of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca 2÷ binding proteins; (d) the free [Ca z÷] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca 2÷ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca 2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+]. T HE intracellular rapidly exchanging stores of Ca 2+have attracted increasing interest during the last several years (for reviews see Carafoli, 1987;Berridge, 1993;Pozzan et al., 1994). These structures are responsible for a fundamental step in many types of cell activation, the release of Ca 2+ to the cytosol taking place not at the surface but at multiple sites within the cytoplasm. Among cytoplasmic organelles, the ER is commonly identified as the cytological counterpart of the stores. Results in various cell types have however indicated that not the entire ER, but discrete areas (e.g., the sarcoplasmic reticuAddress correspondence to Prof. T. Pozzan, Dept. of Biomedical Sciences, University of Padova, Via Trieste, 75, 35121 Padova, Italy. Ph.: 39-49-8286568. Fax: 39-4...

The successful expression of organelle-targeted GFP mutants in live eukaryotes expands the uses of this fluorescent protein in cell biology, allowing direct access to key biological issues, such as the study of the interactions of different organelles in vivo. These results also open the way to other exciting applications, such as the direct study of protein redistribution and protein-protein interactions in living cells.

Lysyl hydroxylases catalyze hydroxylation of collagen lysines, and sustain essential roles in extracellular matrix (ECM) maturation and remodeling. Malfunctions in these enzymes cause severe connective tissue disorders. Human lysyl hydroxylase 3 (LH3/PLOD3) bears multiple enzymatic activities, as it catalyzes collagen lysine hydroxylation and also their subsequent glycosylation. Our understanding of LH3 functions is currently hampered by lack of molecular structure information. Here, we present high resolution crystal structures of full-length human LH3 in complex with cofactors and donor substrates. The elongated homodimeric LH3 architecture shows two distinct catalytic sites at the N- and C-terminal boundaries of each monomer, separated by an accessory domain. The glycosyltransferase domain displays distinguishing features compared to other known glycosyltransferases. Known disease-related mutations map in close proximity to the catalytic sites. Collectively, our results provide a structural framework characterizing the multiple functions of LH3, and the molecular mechanisms of collagen-related diseases involving human lysyl hydroxylases.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+Ic, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+Isr) is much higher than in the other compartments (>100 JIM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (-1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2 , the kinetics and amplitudes of the changes in [cationii]sr evoked by the various stimuli could also be directly analyzed. INTRODUCTION Stimulus-contraction (MacLennan et al., 1985;Lytton and MacLennan, 1988;Burk et al., 1989) and the overall molecular architecture of the pump (Toyoshima et al., 1993) as well as the kinetic details of the transport mechanism (Inesi et al., 1992) have been clarified. Similarly, the Ca2+ release channels, also known as the ryanodine receptors (Takeshima et al., 1989;Otsu et al., 1990;Giannini et al., 1992;Sorrentino and Volpe, 1993) (Rizzuto et al., 1992). Through the addition to the cDNA coding for the native photoprotein of specific targeting sequences or through its fusion with suitable polypeptides, we have produced a series of new aequorin chimeras targeted to different intracellular locations, the mitochondrial matrix (Rizzuto et al., 1992, nucleoplasm (Brini et al., 1993nucleoplasm (Brini et al., , 1994 (Franzini-Armstrong et al., 1987) and is thought to play a major role in intralumenal Ca2' binding (Ikemoto et al., 1989;Lytton and Nigam, 1992;Damiani and Margreth, 1994 Primary cultures of skeletal muscle were prepared from newborn rats (2-3 days) as described previously (Cantini et al., 1994). In brief, posterior limb muscles were remove...