In vivo examination of membrane protein localization and degradation with green fluorescent protein.

Hampton, Randolph Y.; Koning, Ann J.; Wright, Robin; Rine, Jasper

doi:10.1073/pnas.93.2.828

Cited by 100 publications

(93 citation statements)

References 11 publications

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“…Degradation of proteins in the ER was analysed both by tagging an ER resident membrane protein [29] and a mutated secretory protein [30]. The trans-Golgi network (TGN), a major sorting station of the secretory pathway, was not only visualized by following a tagged secretory protein [26] but also by fusing GFP to the TGN resident marker protein TGN38.…”

Section: Secretory Pathwaymentioning

confidence: 99%

Green fluorescent protein: applications in cell biology

1996

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The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted. Recent studies where GFP technology has revealed new insights regarding physiological activities of living cells are discussed.

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Section: Secretory Pathwaymentioning

confidence: 99%

Green fluorescent protein: applications in cell biology

1996

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“…Affinitypurified polyclonal antiubiquitin antibody was generously provided by Arthur Haas (Wisconsin Medical College, Milwaukee, WI;Tiemey et al, 1992). All other reagents were obtained as described previously (Hampton and Rine, 1994;Hampton et al, 1996).…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

“…Yeast were grown and transformed using the media, conditions, and techniques described earlier (Hampton and Rine, 1994;Hampton et al, 1996). All experiments were conducted in yeast minimal medium (0.67% yeast nitrogen base; Difco, Detroit, MI) supplemented as needed.…”

Section: Yeast Culture and Strainsmentioning

confidence: 99%

“…We have previously shown that degradation of the HMG-R isozyme Hmg2p of Saccharomyces cerevisisae is regulated by the mevalonate pathway in a manner similar to that observed for mammalian HMG-R (Hampton and Rine, 1994;Hampton et al, 1996). By designing a selection for mutants deficient in the degradation of yeast Hmg2p, we have defined and isolated genes responsible for the degradation of the Hmg2p isozyme and related substrates.…”

Section: Introductionmentioning

confidence: 99%

“…(Scheffner et al, 1993;Bour et al, 1995;Wiertz et al, i 1996 by The American Society for Cell Biology 2029 R.Y. Hampton et al 1996). Signaling cascades can be started or halted by selective degradation (Jabben et al, 1989;Glotzer et al, 1991;Papavassiliou et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

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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.

Hampton

Gardner

Rine

1996

MBoC

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530

534

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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. Hrdlp had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrdlp 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.

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Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria

et al. 1998

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The ability to distinguish and identify specific subcellular compartments is essential to understanding organelle function, biogenesis, and maintenance within cells and to defining protein trafficking pathways. Fluorescent dyes and/or fluorescently labeled lipid derivatives can be used to identify ER, Golgi complex, and mitochondria. Specific conditions for labeling each of these compartments are described.

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In vivo examination of membrane protein localization and degradation with green fluorescent protein.

Cited by 100 publications

References 11 publications

Green fluorescent protein: applications in cell biology

Green fluorescent protein: applications in cell biology

Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria

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