Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.

The function of the prion protein (PrP c ), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrP c on Ca 2+ homeostasis, by analyzing local Ca 2+ fluctuations in cells transfected with PrP c and Ca 2+ -sensitive aequorin chimeras targeted to defined subcellular compartments. In agonist-stimulated cells, the presence of PrP c sharply increases the Ca 2+ concentration of subplasma membrane Ca 2+ domains, a feature that may explain the impairment of Ca 2+ -dependent neuronal excitability observed in TSEs. PrP c also limits Ca 2+ release from the endoplasmic reticulum and Ca 2+ uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrP c paralogue, display opposite effects, which, however, are abolished by the coexpression of PrP c . These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrP c protects, and Doppel sensitizes, cells toward stress conditions.

The function of the prion protein (PrP), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrP on Ca homeostasis, by analysing local Ca fluctuations in cells transfected with PrP and Casensitive aequorin chimeras targeted to defined subcellular compartments. In agoniststimulated cells, the presence of PrP sharply increases the Ca concentration of sub-plasma membrane Ca domains, a feature that may explain the impairment of Ca-dependent neuronal excitability observed in TSEs. PrP also limits Ca release from the endoplasmic reticulum and Ca uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrP paralogue, display opposite effects, which, however, are abolished by the co-expression of PrP. These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrP protects, and Doppel sensitises, cells towards stress conditions. Introduction The cellular prion protein (PrP) is a highly conserved cell surface glycoprotein, particularly expressed in the central nervous system (CNS), with a still unrecognised function. A conformationally modified isoform (PrP) of PrP is the major component of prions, the ethiological agent at the basis of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). TSEs present as sporadic, genetic, and infectious illnesses, and include Creutzfeldt-Jacob disease (CJD) in humans, and bovine spongiform encephalopathy in cattle (Prusiner, 1998). The mechanism of PrP conversion into PrP is not yet elucidated, nor it is clear if the disease progression relates to PrP toxic effect, or to the deprivation of PrP functionality. The most prominent evidence in support of the former hypothesis is that most PrP-knockout mice remain viable, and do not develop spontaneous neurodegeneration (Bueler et al., 1992; Manson et al., 1994), even if the PrP gene is post-natal deleted (Mallucci et al., 2002). An essential role of PrP in cell survival comes, however, from the finding that wild type PrP transgenes abrogate the cerebellar degeneration and late-onset ataxia developed by some PrPknockout lines over expressing a protein, named Doppel (Dpl) (Sakaguchi et al., 1996; Moore et al., 1999, 2001; Li et al., 2000; Rossi et al., 2001). Dpl is normally absent in the CNS of adult animals and resembles truncated PrP; it lacks the copper binding N-terminus (Brown et al., 1997), while is structurally and biochemically similar to PrP carboxyl end (Silverman et

Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.