Structures,functions and molecular evolution of the penta-EF-hand Ca2+-binding proteins

Penta-EF-hand (PEF) proteins comprise a family of Ca(2+)-binding proteins that have five repetitive EF-hand motifs. Among the eight alpha-helices (alpha1-alpha8), alpha4 and alpha7 link EF2-EF3 and EF4-EF5, respectively. In addition to the structural similarities in the EF-hand regions, the PEF protein family members have common features: (i) dimerization through unpaired C-terminal EF5s, (ii) possession of hydrophobic Gly/Pro-rich N-terminal domains, and (iii) Ca(2+)-dependent translocation to membranes. Based on comparison of amino acid sequences, mammalian PEF proteins are classified into two groups: Group I PEF proteins (ALG-2 and peflin) and Group II PEF proteins (Ca(2+)-dependent protease calpain subfamily members, sorcin and grancalcin). The Group I genes have also been found in lower animals, plants, fungi and protists. Recent findings of specific interacting proteins have started to gradually unveil the functions of the noncatalytic mammalian PEF proteins.     

Both ALG-2 and peflin, penta-EF-hand (PEF) proteins, are stabilized by dimerization through their fifth EF-hand regions

ALG-2 (apoptosis-linked gene-2 protein) and peflin are Ca(2+)-binding proteins and belong to the penta-EF-hand (PEF) protein family, which includes calpain, sorcin, and grancalcin. ALG-2 forms either a homodimer or a heterodimer with peflin like other PEF proteins. In this study, we found that the fifth-EF-hand (EF-5) regions of both ALG-2 and peflin are essential for dimerization and their stabilities. Exogenously expressed EF-5-deletion (DeltaEF-5) mutants of ALG-2 and peflin were unstable and were not detected in HEK293 cells by Western blotting. In a pulse--chase experiment, the DeltaEF-5 mutants were rapidly degraded, but they were stabilized by treatment with a proteasome inhibitor, MG132. In MG132-treated cells, DeltaEF-5 mutants were recovered in the insoluble fractions. Transient coexpression of ALG-2 increased the peflin level. These results indicate that the absence of a fifth EF-hand results in rapid degradation by the proteasome. On the other hand, stable expression of exogenous peflin decreased the amount of endogenous peflin. The amount of peflin that can dimerize with ALG-2 seems to be restricted in mammalian cells.     

Peflin and ALG-2, members of the penta-EF-hand protein family, form a heterodimer that dissociates in a Ca2+-dependent manner

Peflin, a newly identified 30-kDa Ca(2+)-binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 (apoptosis-linked gene 2). We prepared a monoclonal antibody against human peflin. The antibody immunoprecipitated a 22-kDa protein as well as the 30-kDa protein from the lysate of Jurkat cells. Western blotting of the immunoprecipitates revealed that the 22-kDa protein corresponds to ALG-2. This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or ALG-2 whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells. Gel filtration of the cytosolic fraction of Jurkat cells revealed co-elution of peflin and ALG-2 in fractions eluting earlier than recombinant ALG-2, further supporting the notion of heterodimerization of the two PEF proteins. Surprisingly, peflin dissociated from ALG-2 in the presence of Ca(2+). Peflin and ALG-2 co-localized in the cytoplasm, but ALG-2 was also detected in the nuclei as revealed by immunofluorescent staining and subcellular fractionation. Peflin was recovered in the cytosolic fraction in the absence of Ca(2+) but in the membrane/cytoskeletal fraction in the presence of Ca(2+). These results suggest that peflin has features common to those of other PEF proteins (dimerization and translocation to membranes) and may modulate the function of ALG-2 in Ca(2+) signaling.     

Evolutionary and physical linkage between calpains and penta-EF-hand Ca2+-binding proteins

The name calpain was historically given to a protease that is activated by Ca(2+) and whose primary structure contains a Ca(2+)-binding penta-EF-hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2-domain-like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain-like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain-7, indicating that the calpain-7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N-terminal region of calpain-7 has a tandem repeat of microtubule-interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca(2+)-binding proteins including ALG-2 that associates with ALIX (an ESCRT-III accessory protein) and TSG101 (an ESCRT-I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein-protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system.     

The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca2+-dependent manner

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.     

Structure of apoptosis-linked protein ALG-2: insights into Ca2+-induced changes in penta-EF-hand proteins

BACKGROUND: The Ca2+ binding apoptosis-linked gene-2 (ALG-2) protein acts as a proapoptotic factor in a variety of cell lines and is required either downstream or independently of caspases for apoptosis to occur. ALG-2 belongs to the penta-EF-hand (PEF) protein family and has two high-affinity and one low-affinity Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1). RESULTS: We present the 2.3 A resolution crystal structure of Ca2+-Ioaded des1-20ALG-2 (aa 21-191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight alpha helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different from that of calpain and grancalcin. This difference can be described as a rigid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cleaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule. CONCLUSIONS: A different relative orientation of the N- and C-terminal halves of des1-20ALG-2 in the presence of Ca2+ and the peptide as compared to other Ca2+loaded PEF proteins changes substantially the shape of the molecule, exposing a hydrophobic patch on the surface for peptide binding and a large cleft near the dimer interface. We postulate that the binding of a Gly/ Pro-rich peptide in the presence of Ca2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins.     

Identification of the P-body component PATL1 as a novel ALG-2-interacting protein by in silico and far-Western screening of proline-rich proteins

ALG-2 (also named PDCD6) is a 22-kDa Ca(2+)-binding protein that belongs to the penta-EF-hand family including calpain small subunit and interacts with various proteins such as ALIX and Sec31A at their specific sites containing an ALG-2-binding motif (ABM) present in their respective Pro-rich region (PRR). In this study, to search for novel ALG-2-interacting proteins, we first performed in silico screening of ABM-containing PRRs in a human protein database. After selecting 17 sequences, we expressed the PRR or full-length proteins fused with green fluorescent protein (GFP) in HEK293T cells and analysed their abilities to bind to ALG-2 by Far-Western blotting using biotinylated ALG-2 as a probe. As a result, we found 10 positive new ALG-2-binding candidates with different degrees of binding ability. For further investigation, we selected PATL1 (alternatively designated Pat1b), a component of the P-body, which is a cytoplasmic non-membranous granule composed of translation-inactive mRNAs and proteins involved in mRNA decay. Interactions between endogenous PATL1 and ALG-2 proteins were demonstrated by a co-immunoprecipitation assay using their specific antibodies. Furthermore, in immunofluorescence microscopic analyses, PATL1 as well as DCP1A, a well-known P-body marker, co-localized with a subset of ALG-2. This is the first report showing interaction of ALG-2 with a P-body component.     

Structural basis for Ca2+ -dependent formation of ALG-2/Alix peptide complex: Ca2+/EF3-driven arginine switch mechanism

ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.     

Biochemical characterization of a truncated penta-EF-hand Ca2+ binding protein from maize

Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca2+ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension.     

Crystal structure of human grancalcin, a member of the penta-EF-hand protein family

Grancalcin is a Ca(2+)-binding protein expressed at high level in neutrophils. It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca(2+)-dependent manner. Prototypic members of this family are Ca(2+)-binding domains of calpain. Our recent finding that grancalcin interacts with L-plastin, a protein known to have actin bundling activity, suggests that grancalcin may play a role in regulation of adherence and migration of neutrophils. The structure of human grancalcin has been determined at 1.9 A resolution in the absence of calcium (R-factor of 0.212 and R-free of 0.249) and at 2. 5 A resolution in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices and only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the unpaired EF5 hands in a manner similar to that observed in calpain, confirming this mode of association as a paradigm for the PEF family. Only one Ca(2+) was found per dimer under crystallization conditions that included CaCl(2). This cation binds to EF3 in one molecule, while this site in the second molecule of the dimer is unoccupied. This unoccupied site shows higher mobility. The structure determined in the presence of calcium, although does not represent a fully Ca(2+)-loaded form, suggests that calcium induces rather small conformational rearrangements. Comparison with calpain suggests further that the relatively small magnitude of conformational changes invoked by calcium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion that calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character.     

Identification of Alix-type and Non-Alix-type ALG-2-binding sites in human phospholipid scramblase 3: differential binding to an alternatively spliced isoform and amino acid-substituted mutants

ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca(2+)-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2(DeltaGF122), pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2(DeltaGF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca(2+)-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(DeltaGF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2(DeltaGF122): (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.     

The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctate distribution of the amino-terminal truncation form of Alix/AIP1

ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca(2+)-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca(2+) with FLAG-tagged Alix or FLAG-tagged Alix Delta EBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged Alix Delta ABS, another mutant lacking the region comprising amino acids 798-841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca(2+)-dependent fashion, whereas FLAG-tagged ALG-2(E47A/E114A), a Ca(2+)-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca(2+). Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCT Delta ABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca(2+)-dependent interaction with Alix.     

Structure and function of ALG-2, a penta-EF-hand calcium-dependent adaptor protein

ALG-2 (a gene product of PDCD6) is a 22-kD protein containing five serially repetitive EF-hand structures and belongs to the penta-EF-hand (PEF) family, including the subunits of typical calpains. ALG-2 is the most conserved protein among the PEF family members and its homologs are widely found in eukaryotes. X-ray crystal structures of various PEF proteins including ALG-2 have common features: presence of eight α-helices and dimer formation via paired EF5s that are positioned in anti-parallel orientation. ALG-2 forms a homodimer and a heterodimer with its closest paralog peflin. Like calmodulin, a well-known four-EF-hand protein, ALG-2 interacts with various proteins in a Ca²⁺-dependent fashion, but the binding motifs are completely different. With some exceptions, ALG-2-interacting proteins commonly contain Pro-rich regions, and ALG-2 recognizes at least two distinct Pro-containing motifs: PPYP(X)nYP (X, variable; n=4 in ALIX and PLSCR3) and PXPGF (represented by Sec31A). A shorter alternatively spliced isoform, lacking two residues and designated ALG-2^ΔGF122, does not bind ALIX but maintains binding capacity to Sec31A. X-ray crystal structural analyses have revealed that binding of calcium ions induces the configuration of the side chain of R125 so that it opens Pocket 1, which accepts PPYP, but Pocket 1 remains closed in the case of ALG-2^ΔGF122. ALG-2 dimer has two ligand-binding sites, each in a monomer molecule, and appears to function as a Ca²⁺-dependent adaptor protein to either stabilize a preformed complex or to bridge two proteins on scaffolds in systems of the endosomal sorting complex required for transport (ESCRT) and ER-to-Golgi transport.     

Heterogeneous nuclear ribonucleoprotein K interacts with and is proteolyzed by calpain in vivo

Calpain is a cytosolic "modulator protease" that modulates cellular functions in response to Ca2+. To identify in vivo substrates of calpain, yeast two-hybrid screening was done using the 5-EF-hand (penta-EF-hand; PEF) domain of the micro-calpain large subunit (domain IV), since several possible in vivo substrates for calpain have been previously reported to bind to the 5-EF-hand domains. Other than the regulatory subunit of calpain, which binds to the domain IV, heterogeneous nuclear ribonucleoproteins (hnRNP) K and R were identified, and shown to be proteolyzed by micro-calpain in vitro. When expressed in COS7 cells, hnRNP K and micro-calpain co-localized in the cytosol, and Ca2+-ionophore stimulation of the cells resulted in proteolysis of hnRNP K, indicating that hnRNP K is an in vivo substrate for calpain. Now, hnRNP K is considered to function as a scaffold protein for its binding proteins, such as PKCdelta and C/EBPbeta, which were reported to be calpain substrates, suggesting that hnRNP-K is a scaffold for calpain to proteolyze these proteins.     

Ca2+ binding to EF hands 1 and 3 is essential for the interaction of apoptosis-linked gene-2 with Alix/AIP1 in ocular melanoma

Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca(2+)-binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca(2+) concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca(2+) concentration. However, binding of Ca(2+) to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca(2+) affinity. The addition of N-terminal ALG-2 peptides, residues 1-22 or residues 7-17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca(2+) binding to EF-3, while Ca(2+) binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition.     

The PEF family proteins sorcin and grancalcin interact in vivo and in vitro

The penta-EF hand (PEF) family of calcium binding proteins includes grancalcin, peflin, sorcin, calpain large and small subunits as well as ALG-2. Systematic testing of the heterodimerization abilities of the PEF proteins using the yeast two-hybrid and glutathione S-transferase pull-down assays revealed the new finding that grancalcin interacts strongly with sorcin. In addition, sorcin and grancalcin can be co-immunoprecipitated from lysates of human umbilical vein endothelial cells. Our results indicate that heterodimerization, in addition to differential interactions with target proteins, might be a way to regulate and fine tune processes mediated by calcium binding proteins of the penta-EF hand type.     

Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca(2+)-binding proteins in Dictyostelium discoideum

During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca(2+)-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca(2+)-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca(2+)-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind (45)Ca(2+) and interact with CBP1 in vitro in a Ca(2+)-dependent manner.     

The Ca2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.     

Characterization of two recombinant Drosophila calpains. CALPA and a novel homolog, CALPB.

We have sequenced the cDNA of a novel Ca(2+)-activated cysteine proteinase (calpain) from the fruit fly, Drosophila melanogaster. The predicted protein, designated as CALPB, shows high similarity to the previously identified Drosophila calpain, CALPA. The two proteins were expressed in Escherichia coli and purified to homogeneity by metal-chelate affinity chromatography either from inclusion bodies or from the bacterial cytosol. Both enzymes were Ca(2+)-dependent proteinases and attained half-maximal activation in the presence of millimolar Ca(2+). The activity and the rate of activation of CALPA, but not CALPB, could be activated by phosphatidylinositol 4,5-diphosphate, phosphatidylinositol 4-monophosphate, phosphatidylinositol, and phosphatidic acid. A truncated form of CALPA, lacking the CALPA-specific unique insertion region, has also been expressed and characterized. Although it lacked the 16-amino acid long putative membrane-anchoring segment, its activation by phospholipids was similar to that of the full-length CALPA protein. The enzymes undergo N-terminal autolysis in a Ca(2+)-dependent manner which was shown with CALPB to run parallel with enzyme activation. Moreover, fully autolyzed CALPB lacked the characteristic activation phase indicating the requirement for autolysis upon activation of this calpain form in vitro. The analysis of the mechanism of activation in Drosophila calpains seems to corroborate the autolysis model of calpain activation.