C-terminal Green Fluorescent Protein Research Articles

Cell Surface Expression of α1D-Adrenergic Receptors Is Controlled by Heterodimerization with α1B-Adrenergic Receptors

alpha(1)-Adrenergic receptors (ARs) belong to the large Class I G protein-coupled receptor superfamily and comprise three subtypes (alpha(1A), alpha(1B), and alpha(1D)). Previous work with heterologously expressed C-terminal green fluorescent protein (GFP)-tagged alpha(1)-ARs showed that alpha(1A)- and alpha(1B)-ARs localize to the plasma membrane, whereas alpha(1D)-ARs accumulate intracellularly. We recently showed that alpha(1D)- and alpha(1B)-ARs form heterodimers, whereas alpha(1D)- and alpha(1A)-ARs do not. Here, we examined the role of heterodimerization in regulating alpha(1D)-AR localization using both confocal imaging of GFP- or CFP-tagged alpha(1)-ARs and a luminometer-based surface expression assay in HEK293 cells. Co-expression with alpha(1B)-ARs caused alpha(1D)-ARs to quantitatively translocate to the cell surface, but co-expression with alpha(1A)-ARs did not. Truncation of the alpha(1B)-AR extracellular N terminus or intracellular C terminus had no effect on surface expression of alpha(1D)-ARs, suggesting primary involvement of the hydrophobic core. Co-transfection with an uncoupled mutant alpha(1B)-AR (Delta12alpha(1B)) increased both alpha(1D)-AR surface expression and coupling to norepinephrine-stimulated Ca(2+) mobilization. Finally, GFP-tagged alpha(1D)-ARs were not detected on the cell surface when expressed in rat aortic smooth muscle cells that express no endogenous ARs, but were almost exclusively localized on the surface when expressed in DDT(1)MF-2 cells, which express endogenous alpha(1B)-ARs. These studies demonstrate that alpha(1B)/alpha(1D)-AR heterodimerization controls surface expression and functional coupling of alpha(1D)-ARs, the N- and C-terminal domains are not involved in this interaction, and that alpha(1B)-AR G protein coupling is not required. These observations may be relevant to many other Class I G protein-coupled receptors, where the functional consequences of heterodimerization are still poorly understood.

An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody

Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins.

RNA-binding protein Csx1 mediates global control of gene expression in response to oxidative stress.

Fission yeast Spc1 (Sty1), a stress-activated mitogen-activated protein kinase (MAPK) homologous to human p38, orchestrates global changes in gene expression in response to diverse forms of cytotoxic stress. This control is partly mediated through Atf1, a transcription factor homologous to human ATF2. How Spc1 controls Atf1, and how the cells tailor gene expression patterns to different forms of stress, are unknown. Here we describe Csx1, a novel protein crucial for survival of oxidative but not osmotic stress. Csx1 associates with and stabilizes atf1+ mRNA in response to oxidative stress. Csx1 controls expression of the majority of the genes induced by oxidative stress, including most of the genes regulated by Spc1 and Atf1. These studies reveal a novel mechanism controlling MAPK-regulated transcription factors and suggest how gene expression patterns can be customized to specific forms of stress. Csx1-like proteins in humans may perform similar tasks.

Cellugyrin and Synaptogyrin Facilitate Targeting of Synaptophysin to a Ubiquitous Synaptic Vesicle-sized Compartment in PC12 Cells

Cellugyrin represents a ubiquitously expressed four-transmembrane domain protein that is closely related to synaptic vesicle protein synaptogyrin and, more remotely, to synaptophysin. We report here that, in PC12 cells, cellugyrin is localized in synaptic-like microvesicles (SLMVs), along with synaptogyrin and synaptophysin. Upon overexpression of synaptophysin in PC12 cells, it is localized in rapidly sedimenting membranes and practically is not delivered to the SLMVs. On the contrary, the efficiency of the SLMV targeting of exogenously expressed cellugyrin and synaptogyrin is high. Moreover, expression of cellugyrin (or synaptogyrin) in PC12 cells dramatically and specifically increases SLMV targeting of endogenous synaptophysin. Finally, we utilized the SLMV purification scheme on a series of non-neuroendocrine cell types including the mouse fibroblast cell line 3T3-L1, the Chinese hamster ovary cell line CHO-K1, and the monkey kidney epithelial cell line COS7 and found that a cellugyrin-positive microvesicular compartment was present in all cell types tested. We suggest that synaptic vesicles have evolved from cellugyrin-positive ubiquitous microvesicles and that neuroendocrine SLMVs represent a step along that pathway of evolution.

Sugar transport by mammalian members of the SLC26 superfamily of anion-bicarbonate exchangers.

The mammalian cochlea contains a population of outer hair cells (OHCs) whose electromotility depends on an assembly of 'motor' molecules in the basolateral membrane of the cell. Named 'prestin', the molecule is a member of the SLC26 anion transporter superfamily. We show both directly and indirectly that SLC26A5, rat prestin, takes up hexoses when expressed in several cell lines. Direct measurements of labelled fructose transport into COS-7 cells expressing prestin are reported here. Indirect measurements, using imaging techniques, show that transfected HEK-293 or CHO-Ki cells undergo reversible volume changes when exposed to isosmotic glucose-fructose exchange. The observations are consistent with the sugar transport. A similar transport was observed using a C-terminal green fluorescent protein (GFP)-tagged pendrin (SLC26A4) construct. Cells transfected with GFP alone did not respond to sugars. The data are consistent with fructose being transported by prestin with an apparent Km=24 nm. From the voltage-dependent capacitance of transfected cells, we estimate that 250,000 prestin molecules were present and hence that the single transport rate is not more than 3000 fructose molecules s(-1). Comparison of the transfected cell swelling rates induced by fructose and by osmotic steps indicates that water was co-transported with sugar. We suggest that the structure of SLC26 family members allows them to act as neutral substrate transporters and may explain observed properties of cochlear hair cells.

Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar.

NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa x Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue.

Stable Polarized Expression of hCAT-1 in an Epithelial Cell Line

Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-[3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-[3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization.

DNA polymerase eta undergoes alternative splicing, protects against UV sensitivity and apoptosis, and suppresses Mre11-dependent recombination.

Polymerase eta (pol eta) is a low-fidelity DNA polymerase that is the product of the gene, POLH, associated with the human XP variant disorder in which there is an extremely high level of solar-induced skin carcinogenesis. The complete human genomic sequence spans about 40 kb containing 10 coding exons and a cDNA of 2.14 kb; exon I is untranslated and is 6 kb upstream from the first coding exon. Using bacterial artificial chromosomes (BACs), the gene was mapped to human chromosome band 6p21 and mouse band 17D. The gene is expressed in most tissues, except for very low or undetectable levels in peripheral lymphocytes, fetal spleen, and adult muscle; exon II, however, is frequently spliced out in normal cells and in almost half the transcripts in the testis and fetal liver. Expression of POLH in a multicopy episomal vector proved nonviable, suggesting that overexpression is toxic. Expression from chromosomally integrated linear copies using either an EF1-alpha or CMV promoter was functional, resulting in cell lines with low or high levels of pol eta protein, respectively. Point mutations in the center of the gene and in a C-terminal cysteine and deletion of exon II resulted in inactivation, but addition of a terminal 3 amino acid C-terminal tag, or an N- or C-terminal green fluorescent protein, had no effect on function. A low level of expression of pol eta eliminated hMre11 recombination and partially restored UV survival, but did not prevent UV-induced apoptosis, which required higher levels of expression. Polymerase eta is therefore involved in S-phase checkpoint and signal transduction pathways that lead to arrest in S, apoptosis, and recombination. In normal cells, the predominant mechanism of replication of UV damage involves pol eta-dependent bypass, and Mre11-dependent recombination that acts is a secondary, backup mechanism when cells are severely depleted of pol eta.

M Phase-Specific Association of Human Topoisomerase IIIβ with Chromosomes

Two isoforms, 1 and 2, of human DNA topoisomerase IIIβ were expressed in HeLa cells as a fusion protein to the C-terminus of green fluorescent protein (GFP). The fusion protein of the isoform 1 was found to be localized to the nucleus, and to be associated with chromosomes during metaphase and anaphase. As yeast top3 mutants are known to exhibit phenotypes indicative of defective chromosome segregation, the result suggests that the isoform 1 of the human enzyme may also be involved in chromosome segregation. Two-hybrid screening for interaction partners of the isoform identified three candidate genes: CENP-F, a gene encoding a centromere protein and two genes of no known function, one of which was novel. The GFP fusion of the isoform 2 was found in the cytoplasm, indicating the nuclear localization signal sequence in the isoform 1 is in the C-terminal part that is different between the two isoforms.

A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74-GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74-GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74-GFP chimeras in recombinant baculoviruses. When C-terminal region S580-F645 was deleted from p74, p74-GFP chimera localization became non-specific and chimeras became soluble. p74 region S580-F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.

Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.

As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For > 80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.

Green fluorescent protein-tagged sarco(endo)plasmic reticulum Ca2+-ATPase overexpression in Paramecium cells: isoforms, subcellular localization, biogenesis of cortical calcium stores and functional aspects.

We have followed the time-dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP) attached to the C-terminus of the PtSERCA1 gene. The outlines of alveolar sacs (ASs) are labelled, as is the endoplasmic reticulum (ER) throughout the cell. When GFP fluorescence is compared with previous anti-PtSERCA1 antibody labelling, the much wider distribution of GFP (ER+ASs) indicates that only a small amount of SERCA molecules is normally retained in the ER. A second isoform, PtSERCA2, also occurs and its C-terminal GFP-tagging results in the same distribution pattern. However, when GFP is inserted in the major cytoplasmic loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, probably because of the presence of the overt C-terminal KKXX ER-retention signal and/or masking of a signal for transfer into ASs. On the overall cell surface, new SERCA molecules seem to be permanently delivered from the ER to ASs by vesicle transport, whereas in the fission zone of dividing cells ASs may form anew. In cells overexpressing PtSERCA1 (with C-terminal GFP) in ASs, [Ca2+]i regulation during exocytosis is not significantly different from controls, probably because their Ca2+ pump has to mediate only slow reuptake.

Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames.

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases.

Insertion of light-harvesting chlorophyll a/b protein into the thylakoid topographical studies.

The major light-harvesting chlorophyll a/b-binding protein (Lhcb1,2) of photosystem II is inserted into the thylakoid via the signal recognition particle dependent pathway. However, the mechanism by which the protein enters the membrane is at this time unknown. In order to define some topographical restrictions for this process, we constructed several recombinant derivatives of Lhcb1 carrying hexahistidine tags at either protein terminus or in the stromal loop domain. Additionally, green fluorescent protein (GFP) was fused to either terminus. None of the modifications significantly impair the pigment-binding properties of the protein in the in vitro reconstitution of LHCII. With the exception of the C-terminal GFP fusion, all mutants stably insert into isolated thylakoids in the absence of Ni2+ ions. The addition of low concentrations of Ni2+ ions abolishes the thylakoid insertion of C-terminally His-tagged mutants whereas the other His-tagged proteins fail to insert only at higher Ni2+ concentrations. The C-terminus of Lhcb1 must cross the membrane during protein insertion whereas the other sites of Lhcb1 modification are positioned on the stromal side of LHCII. We conclude that a Ni2+-complexed His tag and fusion to GFP inhibit translocation of the protein C-terminus across the thylakoid. Our observations indicate that the N-terminal and stromal domain of Lhcb1 need not traverse the thylakoid during protein insertion and are consistent with a loop mechanism in which only the C-terminus and the lumenal loop of Lhcb1 are translocated across the thylakoid.

ADP-ribosylation factor (ARF)-like 4, 6, and 7 represent a subgroup of the ARF family characterized by rapid nucleotide exchange and a nuclear localization signal

The novel ARF-like GTPase ARL7 is a close relative of ARL4 and ARL6 (71% and 59% identical amino acids). A striking characteristic of these GTPases is their basic C-terminus which, when fused to the C-terminus of green fluorescent protein (GFP), targets the constructs to the nucleus of transfected COS-7 cells. Full length ARL4 was detected in both nuclear and extranuclear compartments, whereas a construct of ARL4 lacking its C-terminus was excluded from the nucleus. Nucleotide exchange rates of recombinant ARL4, ARL6 and ARL7 were similar and appeared considerably higher than those of other members of the ARF family (ARF1, ARP). It is concluded that ARL4, ARL6 and ARL7 form a subgroup within the ARF family with similar, possibly nuclear, function.

BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons.

The protein family of mammalian neurotrophins, comprising nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and -4/5 (NT-3, NT-4/5), supports the survival and the phenotype of neurons from the central as well as the peripheral nervous system (CNS, PNS). In addition, exogenous application of neurotrophins has recently been found to modulate synaptic transmission in the rodent CNS. However, to provide evidence for a role of neurotophins as endogenous fast acting modulators of synaptic transmission, the synaptic localization and secretion of neurotrophins needs to be shown. We have now constructed a fusion protein consisting of N-terminal BDNF (the most abundant neurotrophin in the rodent hippocampus and neocortex) and C-terminal green fluorescent protein (GFP) to elucidate the cellular localization of BDNF in cortical neurons. Transient expression of BDNF-GFP in COS-7 cells revealed that the cellular localization in the trans-Golgi network (TGN), the processing of precursor proteins and the secretion of mature BDNF-GFP is indistinguishable from the properties of untagged BDNF. Upon transient transfection of primary rat cortical neurons, BDNF-GFP was found in secretory granules of the regulated pathway of secretion, as indicated by colocalization with the secretory granule marker secretogranin II. BDNF-GFP vesicles were found in the neurites of transfected neurons with a pattern reminiscent of the localization of endogenous BDNF in untransfected cortical neurons. BDNF-GFP vesicles were found predominantly in the somatodendritic compartment of the neurons, whereas additional axonal localization was found less frequently. Immunocytochemical staining of synaptic terminals with synapsin I antibodies revealed that the density of BDNF-GFP vesicles is elevated in the vicinity of synaptic junctions, indicating that BDNF is localized appropriately to function as an acute modulator of synaptic transmission. These data suggest that BDNF-GFP will be a useful tool to investigate synaptic release of BDNF during physiological synaptic stimulation, and will thereby allow us to elucidate the participation of neurotrophin release in activity dependent synaptic plasticity.

A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events.

Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low-molecular weight A-kinase Anchoring Protein, called AKAP18, which targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid-modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK-293 cells expressing the cardiac L-type Ca2+ channel promoted a 34 9% increase in cAMP-responsive Ca2+ currents. In contrast, a targeting-deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP-1-mediated insulin secretion in a pancreatic beta cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation.

Identification of a motor protein required for filamentous growth in Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast-like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating-type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP-E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; delta kin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild-type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In delta kin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)-Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles.

Deletion mapping of the Aequorea victoria green fluorescent protein

Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2–232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300–500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.