Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells.

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27 kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys(49) and Cys(71)), which could play a role in this oligomerization. Site-directed mutagenesis of Cys(49) and Cys(71) showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

Role of clathrin in the regulated secretory pathway of pancreatic beta-cells.

The role of clathrin in the sorting of proinsulin to secretory granules, the formation of immature granules and their subsequent maturation is not known. To this end, primary rat pancreatic beta-cells were infected with a recombinant adenovirus co-expressing the Hub fragment, a dominant-negative peptide of the clathrin heavy chain and enhanced green fluorescent protein (EGFP as a marker of infected cells). A population of cells expressing the highest levels of EGFP (and thus Hub) was obtained using a fluorescence-activated cell sorter (FACS). Control cells were infected with an adenovirus expressing EGFP alone. By immunofluorescence, control cells showed intense staining for both clathrin light chain and proinsulin in a perinuclear region. In cells expressing high levels of Hub, the clathrin light-chain signal was faint and diffuse in keeping with its displacement from membranes. There was, however, no detectable effect of Hub expression on proinsulin staining or disposition within the cell. Proinsulin sorting and conversion, and the fate (release and/or degradation) of insulin and C-peptide, was studied by pulse-chase and quantitative reverse phase HPLC. In both Hub-expressing and control cells, >99% of all newly synthesized proinsulin was sorted to the regulated pathway and there was no effect of Hub on proinsulin conversion to insulin. In presence of Hub there was, however, a significant increase in the percentage of C-peptide truncated to des-(27-31)-C-peptide at early times of chase as well as more extensive degradation of C-peptide thereafter. It is concluded that clathrin is not implicated in the sorting or processing of proinsulin or in regulated exocytosis of secretory granules. These results confirm a role for clathrin in the removal of proteases from maturing granules, thus explaining the increased truncation and degradation of C-peptide in cells expressing Hub.

Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells.

AIMS/HYPOTHESIS: Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells. METHODS: SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity. RESULTS: Secreted alkaline phosphatase showed high basal secretion (6.6% total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8% total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion. CONCLUSION/INTERPRETATION: These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both.

Trafficking/sorting and granule biogenesis in the beta-cell.

Proinsulin is packaged into nascent (immature, clathrin-coated) secretory granules in the trans-Golgi network (TGN) of the beta -cell along with other granular constituents including the proinsulin conversion enzymes. It is assumed that such packaging is dependent on an active sorting process, separating granular proteins from other secretory or membrane proteins, but the mechanism remains elusive. As granules mature, the clathrin coat is lost, the intragranular milieu is progressively acidified, and proinsulin is converted to insulin and C-peptide. Loss of clathrin is believed to arise by budding of clathrin-coated vesicles from maturing granules, carrying with them any inappropriate or unnecessary products and providing an additional means for refinement of granular content.

Membrane localization and biological activity of SNAP-25 cysteine mutants in insulin-secreting cells.

The tSNARE SNAP-25 is expressed in pancreatic (beta)-cells and is involved in the regulated release of insulin. It has been shown previously that SNAP-25 associates with the plasma membrane consequent to palmitoylation of one or more cysteines in the central region of the molecule. The importance of palmitolyation in the biological function of SNAP-25 in exocytosis was not addressed. Furthermore, studies on both SNAP-25 and its non-palmitoylated homologues SNAP-29 and sec9, have suggested an alternative or complementary mechanism for membrane association involving interaction with syntaxin. To address these issues, we have now studied the behavior and biological activity of cysteine mutant SNAP-25 in insulin-secreting (HIT) cells. While 91% of native SNAP-25 was associated with the membrane, this value decreased to 56% for the single cysteine mutant C85/A and to 10% for the double (C85,88/A) and quadruple (C85,88,90,92/A) mutants. The mutant SNAP-25 forms were all found to bind syntaxin 1A with equal efficacy. Over-expression of syntaxin 1A in HIT cells allowed for partial relocalization of both the double and quadruple SNAP-25 cys mutants to the membrane. By introducing a further mutation to the SNAP-25 molecules to render them resistant to botulinum neurotoxin E, it was possible to study their ability to reconstitute regulated insulin secretion in toxin-treated HIT cells. Native SNAP-25 was able to fully reconstitute secretory activity in such cells. Despite the fact that the single cysteine mutant was significantly displaced to the cytosol, it still displayed 82% activity in the secretion reconstitution assay, and a similar discrepancy was seen for the double mutant. Even the quadruple mutant with no remaining cysteines was able to support a minimal level of secretion. It is concluded that both palmitoylation and binding to syntaxin are implicated in membrane association of SNAP-25. This as well as the discrepancy between membrane localization and biological activity of the cysteine mutants, suggests a complex, multi-component process for association of SNAP-25 with the membrane and its recruitment to a biologically productive state.

Poly(ADP-ribose) polymerase: structure-function relationship.

Dissection of the human poly(ADP-ribose) polymerase (PARP) molecule in terms of its structure-function relationship has proved to be an essential step towards understanding the biological role of poly(ADP-ribosylation) as a cellular response to DNA damage in eukaryotes. Current approaches aimed at elucidating the implication of this multifunctional enzyme in the maintenance of the genomic integrity will be presented.

Structure and function of poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP) participates in the intricate network of systems developed by the eukaryotic cell to cope with the numerous environmental and endogenous genetoxic agents. Cloning of the PARP gene has allowed the development of genetic and molecular approaches to elucidate the structure and the function of this abundant and highly conserved enzyme. This article summarizes our present knowledge in this field.

Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

The zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc-finger, were overproduced in Escherichia coli, in CV-1 monkey cells or in human fibroblasts to study their DNA-binding properties, the trans-dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA-binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Furthermore, overproduced wild-type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc-finger of PARP for DNA single-stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.

The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity.

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.

Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity.

Bacterially expressed fusion proteins containing the COOH-terminal domain of the human poly(ADP-ribose)polymerase were analyzed by means of a novel assay, the "activity blot," which allows the detection of transferred polypeptides involved in poly(ADP-ribose) synthesis. Deletion analysis demonstrated that the 40-kDa COOH-terminal region of the enzyme is an autonomous catalytic domain exhibiting both the polymerizing and branching activities in the absence of DNA. Site-directed mutagenesis demonstrated that lysine 893 is essential for these catalytic processes. In addition, sequence similarities obtained with the NAD(P)+ amino acid dehydrogenases suggest that (i) lysine 893 may interact with the substrates of poly(ADP-ribose)polymerase and (ii) the COOH-terminal part of the 40-kDa fragment may also contain a Rossman fold structure.

The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA.

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a zinc-binding protein that specifically binds to a DNA strand break in a zinc-dependent manner. We describe here the cloning and expression in Escherichia coli of a cDNA fragment encoding the two putative zinc fingers (FI and FII) domain of the human poly(ADP-ribose) polymerase. Using site-directed mutagenesis, we identified the amino acids involved in metal coordination and analyzed the consequence of altering the proposed zinc-finger structures on DNA binding. Disruption of the metal binding ability of the second zinc finger, FII, dramatically reduced target DNA binding. In contrast, when the postulated Zn(II) ligands of FI were mutated, the DNA binding activity was only slightly affected. DNase I protection studies showed that the FII is involved in the specific recognition of a DNA strand break. These results demonstrate that poly(ADP-ribose) polymerase contains a type of zinc finger that differs from previously recognized classes in terms of both structure and function.

Zinc-binding domain of poly(ADP-ribose)polymerase participates in the recognition of single strand breaks on DNA.

Poly(ADP-ribose)polymerase is a chromatin-associated enzyme of eukaryotic cell nuclei that catalyses the covalent attachment of ADP-ribose units from NAD+ to various nuclear acceptor proteins. This post-translational modification has been postulated to influence several chromatin functions, particularly those where nicking and rejoining of DNA occur. Poly(ADP-ribosyl)ation reactions are strictly dependent upon the presence of interruptions on DNA. We have recently demonstrated that the DNA-binding domain of the protein containing two putative "zinc-fingers" binds DNA in a zinc-dependent manner. The basis for the recognition of the DNA strand breaks by this enzyme, and more precisely, its 29,000 Mr N-terminal part, which contains the metal binding sites, needed to be clarified. DNA probes harbouring a single strand interruption at a defined position were constructed from synthetic oligonucleotides. DNase I protection studies show that poly(ADP-ribose)polymerase specifically binds to a DNA single-strand break by its metal-binding domain depending upon the presence of Zn(II). These results support the idea that the enzyme participates to the maintenance of DNA integrity in eukaryotes.

Poly(ADP-ribose)polymerase: a novel finger protein.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.