The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

The Ubiquitin System

In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.

Protein folding in the cell.

INTRODUCTION .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 DOMAIN ORGANIZATION: The Typical ABC Transporter . . . . . . . . . . . . . . . . . 73 THE TRANSMEMBRANE DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 The "Two-Times-Six" Helix Paradigm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Sequence Similarities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 THE ATP-BINDING DOMAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 PERIPLASMIC-BINDING PROTEINS ... . 84 SUBSTRATE SPECIFICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 THE ROLE OF ATP : Coupling Energy to Transport . . . . . . . . . . . . . . . . . . . " . . . . . 88 COVALENT MODIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 CELLULAR FUNCTIONS OF ABC TRANSPORTERS . . . . . . . . . . . . . . . . . . . . . . . 9 1 Nutrient Uptake . . . . . . . . . . . . ....... . 9 1 Protein Export ....... . .... . . . . . . . . . . . . ... . ......... . ...... . ... . .. 93 Intracellular Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Regulation of ABC Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Regulation by ABC Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Drug and Antibiotic Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Channel Functions: CFTR and P-glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 MECHANISMS OF SOLUTE TRANSLOCATION . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Structure of the Transmembrane Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Channels and Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 I Energy Coupling andlor Gating . 102 CONCLUDING REMARKS . 103

ABC Transporters: From Microorganisms to Man

We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane. Because the channel diameter can accommodate only a single strand of RNA or DNA, each polymer traverses the membrane as an extended chain that partially blocks the channel. The passage of each molecule is detected as a transient decrease of ionic current whose duration is proportional to polymer length. Channel blockades can therefore be used to measure polynucleotide length. With further improvements, the method could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.

/pdf/characterization-of-individual-polynucleotide-molecules-b5ji6o583u.pdf

Characterization of individual polynucleotide molecules using a membrane channel

Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

Small GTP-Binding Proteins

Ran is a nuclear Ras-like GTPase that is required for the bidirectional transport of proteins and ribnucleoproteins across the nuclear pore complex (NPC). A key regulator of the Ran GTP/GDP cycle is the 70-kD Ran-GTPase-activating protein RanGAP1. Here, we report the identification and localization of a novel form of RanGAP1. Using peptide sequence analysis and specific mAbs, RanGAP1 was found to be modified by conjugation to a ubiquitin-like protein. Immunoblot analysis and immunolocalization by light and EM demonstrated that the 70-kD unmodified from of RanGAP1 is exclusively cytoplasmic, whereas the 90-kD modified form of RanGAP1 is associated with the cytoplasmic fibers of the NPC. The modified form of RanGAP1 also appeared to associated with the mitotic spindle apparatus during mitosis. These findings have specific implications for Ran function and broad implications for protein regulation by ubiquitin-like modifications. Moreover, the variety and function of ubiquitin-like protein modifications in the cell may be more diverse than previously realized.

/pdf/a-novel-ubiquitin-like-modification-modulates-the-pkiaqm13w0.pdf

A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

https://lab.rockefeller.edu/simon/assets/file/Lab%20Publications/1991_-_CELL_-_%20A_protein-conducting_channel_in_the_endoplasmic_reticulum.pdf

A protein-conducting channel in the endoplasmic reticulum.

Nopp 140 shuttles on tracks between nucleolus and cytoplasm

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide-sensitive protein(s). Both factors act synergistically.

/pdf/70-kd-heat-shock-related-protein-is-one-of-at-least-two-3gslqi4pfj.pdf

70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

We have purified peroxisomal membranes from Saccharomyces cerevisiae after induction of peroxisomes in oleic acid-containing media. About 30 distinct proteins could be discerned among the HPLC- and SDS-PAGE-separated proteins of the high salt-extracted peroxisomal membranes. The most abundant of these, Pmp27p, was purified and the corresponding gene PMP27 was cloned and sequenced. Its primary structure is 32% identical to PMP31 and PMP32 of the yeast Candida biodinii (Moreno, M., R. Lark, K. L. Campbell, and M. J. Goodman. 1994. Yeast. 10:1447-1457). Immunoelectron microscopic localization of Pmp27p showed labeling of the peroxisomal membrane, but also of matrix-less and matrix containing tubular membranes nearby. Electronmicroscopical data suggest that some of these tubular extensions might interconnect peroxisomes to form a peroxisomal reticulum. Cells with a disrupted PMP27 gene (delta pmp27) still grew well on glucose or ethanol, but they failed to grow on oleate although peroxisomes were still induced by transfer to oleate-containing media. The induced peroxisomes of delta pmp27 cells were fewer but considerably larger than those of wild-type cells, suggesting that Pmp27p may be involved in parceling of peroxisomes into regular quanta. delta pmp27 cells cultured in oleate-containing media form multiple buds, of which virtually all are peroxisome deficient. The growth defect of delta pmp27 cells on oleic acid appears to result from the inability to segregate the giant peroxisomes to daughter cells.

/pdf/giant-peroxisomes-in-oleic-acid-induced-saccharomyces-57ykzaye5y.pdf

G Blobel

Papers

A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.

A protein-conducting channel in the endoplasmic reticulum.

Nopp 140 shuttles on tracks between nucleolus and cytoplasm

70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p.