ABSTRACT
A multivesicular body is a vesicle-filled endosome that targets proteins to the interior of lysosomes. We have identified a conserved eukaryotic protein, human CHMP1, which is strongly implicated in multivesicular body formation. Immunocytochemistry and biochemical fractionation localize CHMP1 to early endosomes and CHMP1 physically interacts with SKD1/VPS4, a highly conserved protein directly linked to multivesicular body sorting in yeast. Similar to the action of a mutant SKD1 protein, overexpression of a fusion derivative of human CHMP1 dilates endosomal compartments and disrupts the normal distribution of several endosomal markers. Genetic studies in Saccharomyces cerevisiae further support a conserved role of CHMP1 in vesicle trafficking. Deletion of CHM1, the budding yeast homolog of CHMP1, results in defective sorting of carboxypeptidases S and Y and produces abnormal, multi-lamellar prevacuolar compartments. This phenotype classifies CHM1 as a member of the class E vacuolar protein sorting genes. Yeast Chm1p belongs to a structurally-related, but rather divergent family of proteins, including Vps24p and Snf7p and three novel proteins, Chm2p, Chm5p and Chm6p, which are all essential for multivesicular body sorting. These observations identify the conserved CHMP/Chmp family as a set of proteins fundamental to understanding multivesicular body sorting in eukaryotic organisms.
Subject(s)
Alkyl and Aryl Transferases , Nuclear Proteins/physiology , Protein Transport , ATPases Associated with Diverse Cellular Activities , Adaptor Proteins, Signal Transducing , Adenosine Triphosphatases/metabolism , Carboxypeptidases/metabolism , Conserved Sequence , Endosomal Sorting Complexes Required for Transport , Endosomes/metabolism , Humans , Mutation , Repressor Proteins/metabolism , Vacuolar Proton-Translocating ATPases , Vacuoles/physiology , Vesicular Transport Proteins , Yeasts , rab GTP-Binding ProteinsABSTRACT
pRB activates transcription by a poorly understood mechanism that involves relieving negative regulation of the promoter specificity factor Sp1. We show here that MDM2 inhibits Sp1-mediated transcription, that MDM2 binds directly to Sp1 in vitro as well as in vivo, and that MDM2 inhibits the DNA-binding activity of Sp1. Forced expression of pRB relieves MDM2-mediated repression, and interaction of pRB with the MDM2-Sp1 complex releases Sp1 and restores DNA binding. These results suggest a model in which the opposing activities of MDM2 and pRB regulate Sp1 DNA-binding and transcriptional activity.
Subject(s)
Nuclear Proteins , Proto-Oncogene Proteins/physiology , Retinoblastoma Protein/physiology , Sp1 Transcription Factor/metabolism , 3T3 Cells , Animals , Base Sequence , Binding Sites , DNA Primers , Mice , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Sp1 Transcription Factor/antagonists & inhibitors , Sp1 Transcription Factor/chemistry , Transcriptional Activation/physiology , Zinc/metabolismABSTRACT
Chromosome 3q alterations occur frequently in many types of tumours. In a genetic screen for loci present in rhabdomyosarcomas, we identified an isochromosome 3q [i(3q)], which inhibits muscle differentiation when transferred into myoblasts. The i(3q) inhibits MyoD function, resulting in a non-differentiating phenotype. Furthermore, the i(3q) induces a 'cut' phenotype, abnormal centrosome amplification, aneuploidy and loss of G1 arrest following gamma-irradiation. Testing candidate genes within this region reveals that forced expression of ataxia-telangiectasia and rad3-related (ATR) results in a phenocopy of the i(3q). Thus, genetic alteration of ATR leads to loss of differentiation as well as cell-cycle abnormalities.
Subject(s)
Aneuploidy , Cell Cycle Proteins/genetics , G1 Phase/radiation effects , Multigene Family , MyoD Protein/antagonists & inhibitors , Protein Serine-Threonine Kinases , Ataxia Telangiectasia Mutated Proteins , Cell Division , Chromosomes, Human, Pair 3 , Humans , Isochromosomes , Muscles/cytology , MyoD Protein/physiology , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Tumor Cells, CulturedABSTRACT
Transfer of human chromosome 11, which contains the myoD locus, from primary fibroblasts into 10T1/2 cells results in activation of myoD. In contrast, hybrids that retain human chromosome 11 and additional human chromosomes fail to activate myoD. We show that human chromosome 4 inhibits myoD activation. myoD enhancer/promoter reporter constructs show that repression is at the transcriptional level. Chromosome fragment-containing hybrids localize the repressing activity to the region of 4p that contains the homeobox gene MSX1. MSX1 is expressed in primary human fibroblasts and in 10T1/2 cells containing human chromosome 4, while parental 10T1/2 cells do not express Msx1. Forced expression of Msx1 represses myoD enhancer activity. Msx1 protein binds to the myoD enhancer and likely represses myoD transcription directly. Antisense MSX1 relieves repression mediated by chromosome 4. We conclude that MSX1 inhibits transcription of myoD and that myoD is a target for homeobox gene regulation.
Subject(s)
Genes, Homeobox , MyoD Protein/genetics , Animals , Base Sequence , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 4 , DNA Primers/genetics , DNA, Antisense/genetics , Enhancer Elements, Genetic , Extremities/embryology , Fibroblasts , Gene Expression Regulation, Developmental , Humans , Hybrid Cells , Mice , Molecular Sequence DataABSTRACT
The human cytomegalovirus (CMV) virion glycoprotein gpUL4 (gp48) gene expresses a transcript that contains three AUG codons upstream from the one used to initiate synthesis of the gp48 protein. Previously we reported that the second of these AUG codons, AUG2, was necessary but insufficient for inhibition of downstream translation (M. Schleiss, C. R. Degnin, and A. P. Geballe, J. Virol. 65:6782-6789, 1991). We now demonstrate that the coding information of the upstream open reading frame initiated by AUG2 (uORF2) is critical for the inhibitory signal. Several missense mutations, particularly those involving the carboxy-terminal codons of uORF2, inactivate the inhibitory signal, while mutations that preserve the coding content of uORF2 uniformly retain the inhibitory signal. The uORF2 termination codon is essential for inhibition, but leader sequences further downstream are not critical. Conservation of uORF2 among clinical strains of CMV suggests that uORF2 provides an important function in the CMV infectious cycle. Although these results indicate that the peptide product of uORF2 mediates the inhibitory effect, we demonstrate that the uORF2 signal acts only in cis, and we propose a model of inhibition by the gp48 uORF2 signal.
Subject(s)
Cytomegalovirus/genetics , Gene Expression Regulation, Viral , Genes, Regulator , Open Reading Frames , Protein Biosynthesis , Viral Envelope Proteins/biosynthesis , Amino Acid Sequence , Base Sequence , Cell Line , Codon/genetics , Humans , Molecular Sequence Data , Mutagenesis , Oligodeoxyribonucleotides , Plasmids , Polyribosomes/metabolism , Protein Sorting Signals/biosynthesis , Protein Sorting Signals/genetics , Recombinant Proteins/biosynthesis , Restriction Mapping , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Terminator Regions, Genetic , Transfection , beta-Galactosidase/biosynthesisABSTRACT
Posttranscriptional controls modulate the expression of several human cytomegalovirus genes. Previous studies have shown that one cytomegalovirus gene transcript leader contains AUG codons which inhibit translation of a downstream reading frame. However, two other cytomegalovirus gene transcript leaders of similar structure do not inhibit translation. We have extended these studies to the analysis of the structural glycoprotein gp48, whose predominant transcript contains three upstream AUG codons. The 5' leader of this transcript strongly inhibits downstream translation in fibroblasts. Analyses of deletions and point mutations identify the second upstream AUG codon as an essential component of the inhibitory signal. Other leader sequences, but neither the first nor the third AUG codon, are also required. Intriguingly, the inhibitory signal appears also to depend on the amino acid coding information of the short reading frame associated with the second AUG codon. Insights derived from these studies are germane to understanding the translational regulation of other viral and cellular genes of similar structure.
Subject(s)
Cytomegalovirus/genetics , Gene Expression Regulation, Viral , Glycoproteins/genetics , Protein Biosynthesis , Viral Proteins/genetics , Base Sequence , Cell Line , Chromosome Deletion , Codon , DNA, Viral/genetics , DNA, Viral/isolation & purification , Enhancer Elements, Genetic , Genes, Viral , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Polymerase Chain Reaction , Polyribosomes/metabolism , Reading Frames , Restriction Mapping , Transcription, Genetic , TransfectionABSTRACT
S-Adenosylmethionine decarboxylase (AdoMetDC) activity was elevated 18.8-fold in Swiss 3T3 fibroblasts which were depleted of cellular polyamines by using the inhibitor difluoromethylornithine (DFMO). Although the cellular level of AdoMetDC mRNA and the half-life of active AdoMetDC protein were also increased (4.3- and 1.5-fold respectively), together they could not account for the magnitude of the increase in AdoMetDC activity. These data suggested that the translation of AdoMetDC mRNA must be increased in the polyamine-depleted cells to account fully for the elevation in activity. The cellular distribution of AdoMetDC mRNA was examined in the polyamine-depleted cells, and it was found almost exclusively associated with large polysomes. In contrast, AdoMetDC mRNA in untreated controls was very heterogeneous, with the proportion associated with monosomes equal to that associated with large polysomes. The shift of the AdoMetDC message into large polysomes occurred within 18 h after addition of DFMO to the cultures and could be reversed by adding exogenous putrescine. The effect of polyamine depletion on AdoMetDC translation was specific, since there was no change in the distribution in polysomes of either actin mRNA or the translationally controlled mRNA encoding ribosomal protein S16 in the DFMO-inhibited cells. Thus the translational efficiency of AdoMetDC mRNA in vivo is regulated either directly or indirectly by the concentration of intracellular polyamines through a mechanism involving translational initiation, which results in a change in the number of ribosomes associated with this mRNA.
