Systemic and neurologic abnormalities distinguish the lysosomal disorders sialidosis and galactosialidosis in mice.
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Neuraminidase initiates the hydrolysis of sialo-glycoconjugates by removing their terminal sialic acid residues. In humans, primary or secondary deficiency of this enzyme leads to two clinically similar neurodegenerative lysosomal storage disorders: sialidosis and galactosialidosis (GS). Mice nullizygous at the Neu1 locus develop clinical abnormalities reminiscent of early-onset sialidosis in children, including severe nephropathy, progressive edema, splenomegaly, kyphosis and urinary excretion of sialylated oligosaccharides. Although the sialidosis mouse model shares clinical and histopathological features with GS mice and GS patients, we have identified phenotypic abnormalities that seem specific for sialidosis mice. These include progressive deformity of the spine, high incidence of premature death, age-related extramedullary hematopoiesis, and lack of early degeneration of cerebellar Purkinje cells. The differences and similarities identified in these sialidosis and GS mice may help to better understand the pathophysiology of these diseases in children and to identify more targeted therapies for each of these diseases. (+info)
Yeast Ysl2p, homologous to Sec7 domain guanine nucleotide exchange factors, functions in endocytosis and maintenance of vacuole integrity and interacts with the Arf-Like small GTPase Arl1p.
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We previously described the isolation of ysl2-1 due to its genetic interaction with Delta ypt51/vps21, a mutant with a deletion of the coding sequence for the yeast Rab5 homolog, which regulates endocytic traffic between early and late endosomes. Here we report that Ysl2p is a novel 186.8-kDa peripheral membrane protein homologous to members of the Sec7 family. We provide multiple genetic and biochemical evidence for an interaction between Ysl12p and the Arf-like protein Arl1p, consistent with a potential function as an Arf guanine nucleotide exchange factor (GEF). The temperature-sensitive alleles ysl2-307 and ysl2-316 are specifically defective in ligand-induced degradation of Ste2p and alpha-factor and exhibit vacuole fragmentation directly upon a shift to 37 degrees C. In living cells, green fluorescent protein (GFP)-Ysl2p colocalizes with endocytic elements that accumulate FM4-64. The GFP-Ysl2p staining is sensitive to a mutation in VPS27 resulting in the formation of an aberrant class E compartment, but it is not affected by a sec7 mutation. Consistent with the idea that Ysl2p and Arl1p have closely related functions, Delta arl1 cells are defective in endocytic transport and in vacuolar protein sorting. (+info)
Identification of a CTL4/Neu1 fusion transcript in a sialidosis patient.
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The deficiency of the lysosomal neuraminidase (NEU1; sialidase) causes the storage disorder sialidosis with symptoms ranging from eye abnormalities and neurological disturbances to skeletal malformations, mental retardation and early death. Sialidosis patients encompassing a wide spectrum of clinical symptoms were screened for mutations in neu1. We identified the same homozygous interstitial deletion (11 kb) in two patients causing the fusion of exon 10 of CTL4 (New Gene 22; NG22) with the 3'-UTR of neu1. In one patient we found the resulting CTL4/Neu1 fusion transcript, in the other we detected an alternatively spliced CTL4 transcript (retention of intron 9). (+info)
Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y.
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Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket. Furthermore, C341G and C341S mutants had significantly higher k(cat) values with substrates containing the hydrophobic P1 side chain than C341V or C341F. This indicates that the nonhydrophobic property conferred by Gly or Ser gives flexibility or instability to the S1 pocket, which contributes to the increased k(cat) values of C341G or C341S. The results suggest that Cys341 may interact with His397 during catalysis. Therefore, we propose a dual role for Cys341: (a) its hydrophobicity allows it to participate in the formation of the Michaelis complex with hydrophobic substrates, where it maintains an unfavorable steric constraint in the S1 subsite; (b) its interaction with the imidazole ring of His397 contributes to the rate enhancement by stabilizing the tetrahedral intermediate in the transition state. (+info)
Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae.
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The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process. (+info)
Increased proteolytic susceptibility of carboxypeptidase Y caused by modification of the disulfide zipper.
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To investigate the structural importance of a "disulfide zipper" motif of carboxypeptidase Y, disulfide-deficient mutant enzymes were expressed in two strains of Saccharomyces cerevisiae. The mutant enzymes were rapidly degraded into fragments by intracellular proteases. Thus, it is concluded that the disulfide zipper is essential in maintaining the structural integrity of CPase Y against proteolytic susceptibility. (+info)
Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition.
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Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined In in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin-oligonucleotide conjugates (1 to 44 nt with 3'-puromycin) In a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficiently (IC50 < 50 microM). This observation stands in contrast with work on isolated large ribosomal subunits, which Indicates that many of the puromycin-oligonucleotide conjugates we studied should have higher affinity for the peptidyl transferase center than puromycin itself. Two tRNA(Al)-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC50 = 40-56 microM). However, the puromycin minihelices inhibit translation by sequestering one or more soluble translation factors, and do not appear to participate in detectable peptide bond formation with the nascent chain. In contrast, puromycin and other short derivatives act in a factor-independent fashion at the peptidyl transferase center and readily become conjugated to the nascent protein chain. However, even for the short derivatives, much of the translation inhibition occurs without peptide bond formation between puromycin and the nascent chain, a revision of the classical model for puromycin function. This peptide bond-independent mode is likely a combination of multiple effects including inhibition of initiation and failure to properly recycle translation complexes that have reacted with puromycin. (+info)
Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p.
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Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association. (+info)