ABSTRACT
There has recently been considerable progress in understanding the regulation of clathrin-coated vesicle (CCV) formation and function. These advances are due to the determination of the structure of a number of CCV coat components at molecular resolution and the identification of novel regulatory proteins that control CCV formation in the cell. In addition, pathways of (a) phosphorylation, (b) receptor signaling, and (c) lipid modification that influence CCV formation, as well as the interaction between the cytoskeleton and CCV transport pathways are becoming better defined. It is evident that although clathrin coat assembly drives CCV formation, this fundamental reaction is modified by different regulatory proteins, depending on where CCVs are forming in the cell. This regulatory difference likely reflects the distinct biological roles of CCVs at the plasma membrane and trans-Golgi network, as well as the distinct properties of these membranes themselves. Tissue-specific functions of CCVs require even more-specialized regulation and defects in these pathways can now be correlated with human diseases.
Subject(s)
Carrier Proteins/metabolism , Clathrin-Coated Vesicles/physiology , Clathrin/chemistry , Coated Pits, Cell-Membrane/metabolism , Membrane Proteins/metabolism , Adaptor Proteins, Vesicular Transport , Animals , Cell Polarity/physiology , Clathrin/metabolism , Clathrin-Coated Vesicles/metabolism , Cytoskeleton/metabolism , Endocytosis/physiology , Humans , Models, Molecular , Muscle, Skeletal/metabolism , Neurons/metabolism , Protein Transport/physiologyABSTRACT
The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.
Subject(s)
Endoplasmic Reticulum/metabolism , H-2 Antigens/biosynthesis , Immediate-Early Proteins/biosynthesis , Immunodominant Epitopes/biosynthesis , Muromegalovirus/immunology , Muscle Proteins , Peptides/metabolism , Protein Precursors/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Animals , Antigen Presentation , Autoantigens , Biological Transport, Active/immunology , Cell Cycle Proteins , Cell Line , Cysteine Endopeptidases/metabolism , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/immunology , H-2 Antigens/metabolism , Histocompatibility Antigen H-2D , Humans , Immediate-Early Proteins/metabolism , Immunodominant Epitopes/metabolism , Mice , Microsomes/metabolism , Molecular Sequence Data , Molecular Weight , Multienzyme Complexes/metabolism , Peptides/chemical synthesis , Proteasome Endopeptidase Complex , Protein Precursors/chemical synthesis , Protein Precursors/metabolism , Proteins/pharmacology , RatsABSTRACT
We have studied the consequences of heat shock on 20S/26S proteasome activity and activation, the proteasomal subunit composition, proteasome assembly, subunit mRNA stability as well as on the intracellular distribution of proteasomes. Our data show that heat shock locks 20S proteasomes in their latent inactive state and impairs further activation of the 26S proteasome by ATP. Proteasome mRNA levels are decreased after heat shock and the assembly of the proteasome complex is inhibited. Heat shock also induces a rapid reorganisation of the cellular distribution of the proteasome which appears to be connected with proteasome activity and the change of the cellular architecture after heat shock.
Subject(s)
Cysteine Endopeptidases/metabolism , Heat-Shock Response/physiology , Multienzyme Complexes/metabolism , Peptide Hydrolases/metabolism , Adenosine Triphosphate/physiology , Animals , Biotransformation , Catalysis , Cells, Cultured , Cysteine Endopeptidases/isolation & purification , Drosophila/metabolism , Electrophoresis, Polyacrylamide Gel , Eukaryotic Cells/metabolism , Multienzyme Complexes/isolation & purification , Peptide Hydrolases/isolation & purification , Proteasome Endopeptidase Complex , RNA, Messenger/biosynthesisABSTRACT
We have cloned the mouse PA28 proteasome activator cDNAs. Northern blot demonstrates high PA28 mRNA levels in liver, kidney and lung. mRNA levels are low in thymus, spleen and brain. In contrast, PA28 protein levels vary little between these tissues. Immunocytological analysis and cell fractionation experiments demonstrate that both subunits are almost equally distributed between the cytoplasm and the nucleus. Interestingly, PA28alpha spares nucleoli, while PA28beta is strongly enhanced in the nucleolus. This indicates for the first time that the PA28alpha and PA28beta subunits may serve nuclear functions which may be different from and independent of each other.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Proteins/genetics , Amino Acid Sequence , Animals , Autoantigens , Blotting, Northern , Brain/metabolism , Cells, Cultured , Cloning, Molecular , Fluorescent Antibody Technique, Indirect , Immunohistochemistry , Kidney/metabolism , Liver/metabolism , Lung/metabolism , Mice , Molecular Sequence Data , Proteasome Endopeptidase Complex , Proteins/metabolism , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Spleen/metabolism , Thymus Gland/metabolism , Tissue DistributionABSTRACT
The 20S proteasome is widely viewed at as a cytoplasmic multicatalytic proteinase complex: immunocytochemical investigations, however, show that proteasomes are localized in the cytoplasm as well as in the nucleus within the same cell. Strong nuclear accumulation of proteasomes is observed in rapidly dividing cells such as in the early stages of Drosophila embryogenesis and in tumorigenic cells. In fact, dependent on the metabolic state of a certain tissue or cell type its cellular distribution appears differentially regulated. Several of the proteasomal alpha-type subunits carry putative nuclear localization signals which may or may not take part in the regulation of the intracellular distribution of 20S proteasomes. We have examined the functional role of the putative nuclear localization signal (NLS) -KKKQKK-in the Drosophila PROS-28.1 subunit by deletion mutagenesis and transfection experiments. Linkage of the putative PROS-28.1 NLS to BSA as reporter protein and in vitro import studies with permeabilized mouse NIH 3T3 cells show that this NLS is able to induce complete translocation of the reporter protein into the cell nucleus. For analysis of the NLS within the 28-kDa subunit, cDNA deletion constructs were cloned into a pSG5 expression vector and transiently transfected into mouse fibroblast cells. Whereas the deletion of the NLS alone resulted only in a slight impairment of subunit transport into the nucleus, removal of the C-terminal 96 amino acid residues abolished nuclear translocation completely.
