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Biomedical subjects

L Pallanck

Publications and source records attributed to L Pallanck.

14 recordsLinked to original sources
PubMed2001

Genetic interaction between shibire and comatose mutations in Drosophila suggest a role for snap-receptor complex assembly and disassembly for maintenance of synaptic vesicle cycling.

NSF is an ATPase required for the fusion of secretory vesicles with plasma membrane. Conditional comatose (Drosophila homolog of N-ethylmaleimide sensitive fusion factor (NSF)) mutations in Drosophila block synaptic transmission at restrictive temperature. Current models hold that NSF-mediated dissociation of SNARE (SNAp REceptor) complexes on mature synaptic vesicles primes them for exocytic release. Paralysis in comt mutants thus reflects defective exocytosis due to buildup of unresolved SNARE complexes. Here, we analyze effects of blocking synaptic vesicle recycling on behavioral, physiological and biochemical phenotypes of comt. Behavioral recovery of comt animals and recovery of comt synapses, as assayed by electroretinograms, after exposure to high temperature is faster if synaptic vesicle recycling is simultaneously blocked using shi(ts) mutants. Concurrently, 7S complex buildup in comt shi double mutants is substantially lower than in comt mutants alone. In addition, we find that 7S complexes can form on presynaptic plasma membrane if NSF is inhibited after synaptic-vesicle depletion. Thus, our experiments demonstrate a need for continuous NSF activity required not only for dissociating cis-SNARE complexes on plasma membrane after exocytosis, but also for maintaining these cis-SNARE complexes in a dissociated state.

Animals↗
PubMed2001

Partitioning of N-ethylmaleimide-sensitive fusion (NSF) protein function in Drosophila melanogaster: dNSF1 is required in the nervous system, and dNSF2 is required in mesoderm.

The N-ethylmaleimide-sensitive fusion protein (NSF) promotes the fusion of secretory vesicles with target membranes in both regulated and constitutive secretion. While it is thought that a single NSF may perform this function in many eukaryotes, previous work has shown that the Drosophila genome contains two distinct NSF genes, dNSF1 and dNSF2, raising the possibility that each plays a specific secretory role. To explore this possibility, we generated mutations in the dNSF2 gene and used these and novel dNSF1 loss-of-function mutations to analyze the temporal and spatial requirements and the degree of functional redundancy between dNSF1 and dNSF2. Results of this analysis indicate that dNSF1 function is required in the nervous system beginning at the adult stage of development and that dNSF2 function is required in mesoderm beginning at the first instar larval stage of development. Additional evidence suggests that dNSF1 and dNSF2 may play redundant roles during embryonic development and in the larval nervous system. Ectopic expression studies demonstrate that the dNSF1 and dNSF2 gene products can functionally substitute for one another. These results indicate that the Drosophila NSF proteins exhibit similar functional properties, but have evolved distinct tissue-specific roles.

Animals↗
PubMed1998

NSF function in neurotransmitter release involves rearrangement of the SNARE complex downstream of synaptic vesicle docking.

The SNARE hypothesis has been proposed to explain both constitutive and regulated vesicular transport in eukaryotic cells, including release of neurotransmitter at synapses. According to this model, a vesicle targeting/docking complex consisting primarily of vesicle- and target-membrane proteins, known as SNAREs, serves as a receptor for the cytosolic N-ethylmaleimide-sensitive fusion protein (NSF). NSF-dependent hydrolysis of ATP disassembles the SNARE complex in a step postulated to initiate membrane fusion. While features of this model remain tenable, recent studies have challenged fundamental aspects of the SNARE hypothesis, indicating that further analysis of these components is needed to fully understand their roles in neurotransmitter release. We have addressed this issue by using the temperature-sensitive Drosophila NSF mutant comatose (comt) to study the function of NSF in neurotransmitter release in vivo. Synaptic electrophysiology and ultrastructure in comt mutants have recently defined a role for NSF after docking in the priming of synaptic vesicles for fast calcium-triggered fusion. Here we report that an SDS-resistant neural SNARE complex, composed of the SNARE polypeptides syntaxin, n-synaptobrevin, and SNAP-25, accumulates in comt mutants at restrictive temperature. Subcellular fractionation experiments indicate that these SNARE complexes are distributed predominantly in fractions containing plasma membrane and docked synaptic vesicles. Together with the electrophysiological and ultrastructural analyses of comt mutants, these results indicate that NSF functions to disassemble or otherwise rearrange a SNARE complex after vesicle docking and that this rearrangement is required to maintain the readily releasable pool of synaptic vesicles.

Adenosine Triphosphatases↗
PubMed1995

Distinct roles for N-ethylmaleimide-sensitive fusion protein (NSF) suggested by the identification of a second Drosophila NSF homolog.

The N-ethylmaleimide-sensitive fusion protein (NSF) is a cytoplasmic protein implicated in the fusion of intracellular transport vesicles with their target membranes. NSF is thought to function in the fusion of essentially all types of vesicles, including endoplasmic reticulum, Golgi, and endocytic vesicles, as well as secretory vesicles undergoing regulated fusion (for review see Rothman, J.E. (1994) Nature 372, 55-63). However, little experimental evidence exists to address the possibility that organisms might have multiple NSF proteins serving distinct functions in the same or different cells. We previously cloned a neurally expressed Drosophila homolog, dNSF-1 (Ordway, R.W., Pallanck, L., and Ganetzky, B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5715-5719), and have subsequently identified mutations in this gene that confer an apparent failure of synaptic transmission at elevated temperature (Pallanck, L., Ordway, R.W., and Ganetzky, B. (1995) Nature, 376, 25; Siddiqi, O., and Benzer, S. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3253-3257). Here we report that 1) Drosophila contains a second NSF homolog, termed dNSF-2, that exhibits 84% amino acid identity to dNSF-1, 2) dNSF-1 and dNSF-2 display overlapping but different temporal expression, and 3) multiple transcripts are derived from the dNSF-2 gene. These findings raise the possibility that different NSF gene products serve distinct or overlapping functions with the organism.

Amino Acid Sequence↗
PubMed1995

Functional role of the beta subunit of high conductance calcium-activated potassium channels.

Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, alpha and beta. We have examined the functional contribution of the beta subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both alpha and beta subunits were much more sensitive to activation by voltage and calcium than channels composed of the alpha subunit alone, while expression levels, single-channel conductance, and ionic selectivity appeared unaffected. Channels from oocytes expressing both subunits were sensitive to DHS-I, a potent agonist of native maxi-K channels, whereas channels composed of the alpha subunit alone were insensitive. Thus, alpha and beta subunits together contribute to the functional properties of expressed maxi-K channels. Regulation of co-assembly might contribute to the functional diversity noted among members of this family of potassium channels.

Animals↗
PubMed1994

Neurally expressed Drosophila genes encoding homologs of the NSF and SNAP secretory proteins.

Several lines of investigation have now converged to indicate that the neurotransmitter release apparatus is formed by assembly of cytosolic proteins with proteins of the synaptic vesicle and presynaptic terminal membranes. We are undertaking a genetic approach in Drosophila melanogaster to investigate the functions of two types of cytosolic proteins thought to function in this complex: N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs). We have identified Drosophila homologs of the vertebrate and yeast NSF and SNAP genes. Both Drosophila genes encode polypeptides that closely resemble their vertebrate counterparts and are expressed in the nervous system; neither appears to be in a family of closely related Drosophila genes. These results indicate that the Drosophila NSF and SNAP genes are excellent candidates for mutational analysis of neurotransmitter release.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of human and mouse homologs of the Drosophila calcium-activated potassium channel gene, slowpoke.

Potassium channels play important roles in a wide variety of physiological processes. Although several genes encoding voltage-activated potassium channels have been analyzed at the molecular level, no calcium-activated potassium channel gene has yet been characterized in humans. In an effort to provide the foundation for functional analysis of such polypeptides we report the cloning of mouse and human homologs of the Drosophila melanogaster calcium-activated potassium channel gene, slowpoke. Both the human and mouse genes encode polypeptides that have more than 50% amino acid identifies with their Drosophila counterpart. In addition, like the Drosophila slowpoke gene, both the mouse and human genes generate multiple transcripts by alternative splicing. The human gene maps to chromosome 10 based on the results of polymerase chain reaction analysis of genomic DNA from human-hamster hybrid cell lines. Because calcium-activated potassium channels participate in wide variety of cellular functions including neuromuscular communication, secretion and cellular immunity, their continued analysis promises to have broad biological and medical significance.

Amino Acid Sequence↗
PubMed1992

The anticodon and discriminator base are major determinants of cysteine tRNA identity in vivo.

Mutants of the Escherichia coli initiator tRNA (tRNA(fMet)) have been used to examine the role of the anticodon and discriminator base in in vivo aminoacylation of tRNAs by cysteinyl-tRNA synthetase. Substitution of the methionine anticodon CAU with the cysteine anticodon GCA was found to allow initiation of protein synthesis by the mutant tRNA from a complementary initiation codon in a reporter protein. Sequencing of the protein revealed that cysteine comprised about half of the amino acid at the N terminus. An additional mutation, converting the discriminator base of tRNA(GCAfMet) from A73 to the base present in tRNA(Cys) (U73), resulted in a 6-fold increase in the amount of protein produced and insertion of greater than or equal to 90% cysteine in response to the complementary initiation codon. Substitution of C73 or G73 at the discriminator position led to insertion of little or no cysteine, indicating the importance of U73 for recognition of the tRNA by cysteinyl-tRNA synthetase. Single base changes in the anticodon of tRNA(GCAfMet) containing U73 from GCA to UCA, GUA, GCC, and GCG (changes underlined) eliminated or dramatically reduced cysteine insertion by the mutant initiator tRNA indicating that all three cysteine anticodon bases are essential for specific aminoacylation of the tRNA with cysteine in vivo.

Acylation↗
PubMed1992

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗
PubMed1991

Anticodon-dependent aminoacylation of a noncognate tRNA with isoleucine, valine, and phenylalanine in vivo.

An assay based on the initiation of protein synthesis in Escherichia coli has been used to explore the role of the anticodon in tRNA identity in vivo. Mutations were introduced into the initiator tRNA to change the wild-type anticodon from CAU (methionine) to GAU (isoleucine), GAC (valine), and GAA (phenylalanine), where each derivative differs from the preceding by a single base change in the anticodon (underlined). These changes were sufficient to cause the mutant tRNAs to be aminoacylated by the corresponding aminoacyl-tRNA synthetases based on the amino acid inserted into protein from complementary initiation codons. Construction of additional single base anticodon variants (GUU, GGU, GCC, GUC, GCA, and UAA) showed that all three anticodon bases specify isoleucine and phenylalanine identity and that both the middle and the third anticodon bases are important for valine identity in vivo.

Amino Acyl-tRNA Synthetases↗