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      Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator


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          Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12 ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13 ts strain (mutant in the Sec13p component of COPII), and sec23 ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

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          Aggresomes: A Cellular Response to Misfolded Proteins

          Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.
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            A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

            SD Emr (1995)
            We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.
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              Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

              3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the agents of HMG-R degradation but not the regulators of that degradation.

                Author and article information

                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                20 January 2003
                : 160
                : 2
                : 157-163
                Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294
                Author notes

                Address correspondence to Dr. Elizabeth Sztul, Dept. of Cell Biology, MCLM Room 668, 1530 3rd Ave. South, Birmingham, AL 35294. Tel.: (205) 934-1465. Fax: (205) 975-9131. E-mail: esztul@ 123456uab.edu

                Copyright © 2003, The Rockefeller University Press
                : 15 October 2002
                : 6 December 2002
                : 9 December 2002

                Cell biology
                er sorting; proteasomal degradation; cftr; erad; yeast
                Cell biology
                er sorting; proteasomal degradation; cftr; erad; yeast


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