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Dive into the research topics where Janet L. Burton is active.

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Featured researches published by Janet L. Burton.


The EMBO Journal | 1994

Specific interactions of Mss4 with members of the Rab GTPase subfamily.

Janet L. Burton; M E Burns; Evelina Gatti; G J Augustine; P De Camilli

Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4‐GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co‐precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.


Molecular and Cellular Biology | 2000

Hsl1p, a Swe1p Inhibitor, Is Degraded via the Anaphase-Promoting Complex

Janet L. Burton; Mark J. Solomon

ABSTRACT Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G1, accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations inCDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G1-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1pdb-mut) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCFMet30 in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.


Molecular and Cellular Biology | 2008

Pseudosubstrate Inhibition of the Anaphase-Promoting Complex by Acm1: Regulation by Proteolysis and Cdc28 Phosphorylation

Denis Ostapenko; Janet L. Burton; Ruiwen Wang; Mark J. Solomon

ABSTRACT The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.


The EMBO Journal | 2011

Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

Janet L. Burton; Yong Xiong; Mark J. Solomon

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell‐cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40‐containing APC co‐activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APCCdh1 inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A‐motif that interacts with Cdh1 and together with the DB and KEN box is required for APCCdh1 inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A‐motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APCCdh1 whereas lysine removal from the APC substrate Hsl1 converted it into a potent APCCdh1 inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APCCdh1.


Journal of Biological Chemistry | 1997

An Evolutionarily Conserved Domain in a Subfamily of Rabs Is Crucial for the Interaction with the Guanyl Nucleotide Exchange Factor Mss4

Janet L. Burton; Vladimir I. Slepnev; Pietro De Camilli

Mss4 is a guanine nucleotide exchange factor that specifically binds to, and promotes GDP-GTP exchange on, a subset of the Rab GTPases (Burton, J. L., Burns, M. E., Gatti, E., Augustine, G. J., and De Camilli, P. (1994) EMBO J. 13, 5547-5558). In order to identify the domain(s) of the GTPase that is important for this interaction, protein chimeras were constructed between Rab3a, which binds Mss4, and Rab5a, which does not bind Mss4. We have identified the amino-terminal portion of Rab3a as the Mss4-binding region, with the effector domain being critically required for binding and the flanking regions further enhancing the interaction. Sequence comparisons have revealed that Mss4-binding Rabs share more homology with each other than with Rabs that do not bind Mss4. The region of highest homology between these Rabs, which defines them as members of the same evolutionary branch within the Rab subfamily, coincides with the domain shown here to be critical for Mss4 binding. A mutation in the zinc-binding domain of Mss4 (Mss4 D96H), a region that is highly conserved between Mss4 and its yeast homologue Dss4, completely abolished its property to bind to, and promote GDP-GTP exchange on, Rab3a. Thus, the preservation of the Mss4/Dss4-GTPase interaction appears to have been a critical factor in the evolution of this subset of Rab proteins.


PLOS ONE | 2012

Identification of Anaphase Promoting Complex Substrates in S. cerevisiae

Denis Ostapenko; Janet L. Burton; Mark J. Solomon

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an essential ubiquitin ligase that targets numerous proteins for proteasome-mediated degradation in mitosis and G1. To gain further insight into cellular pathways controlled by APC/CCdh1, we developed two complementary approaches to identify additional APC/CCdh1 substrates in budding yeast. First, we analyzed the stabilities of proteins that were expressed at the same time in the cell cycle as known APC/C substrates. Second, we screened for proteins capable of interacting with the Cdh1 substrate-binding protein in a yeast two-hybrid system. Here we characterize five potential APC/C substrates identified using these approaches: the transcription factors Tos4 and Pdr3; the mRNA processing factor Fir1; the spindle checkpoint protein kinase Mps1; and a protein of unknown function, Ybr138C. Analysis of the degradation motifs within these proteins revealed that the carboxyl-terminal KEN box and D-boxes of Tos4 are important for its interaction with Cdh1, whereas the N-terminal domain of Ybr138C is required for its instability. Functionally, we found that a stabilized form of Mps1 delayed cell division upon mild spindle disruption, and that elevated levels of Ybr138C reduced cell fitness. Interestingly, both Tos4 and Pdr3 have been implicated in the DNA damage response, whereas Mps1 regulates the spindle assembly checkpoint. Thus, the APC/CCdh1-mediated degradation of these proteins may help to coordinate re-entry into the cell cycle following environmental stresses.


Molecular Biology of the Cell | 2015

The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae

Denis Ostapenko; Janet L. Burton; Mark J. Solomon

The accumulation of S-phase cyclins regulates DNA synthesis. In budding yeast, the S-phase cyclin Clb5 is degraded in M phase after ubiquitination by the anaphase-promoting complex (APC). Clb5 accumulation in G1 is promoted by its deubiquitination by the Ubp15-deubiquitinating enzyme, which opposes the action of the APC.


Nature Cell Biology | 2008

Securin' M-phase entry

Mark J. Solomon; Janet L. Burton

The anaphase-promoting complex (APC) mediates the ubiquitination and degradation of key M-phase regulators, including cyclins and the anaphase inhibitor securin. Intriguingly, securin can also inhibit the degradation of cyclin B. This competition between substrates permits the accumulation of enough cyclin to drive entry into M phase.


Cellular Signalling | 2017

Transcriptional and post-transcriptional regulation of Cdc20 during the spindle assembly checkpoint in S. cerevisiae

Ruiwen Wang; Janet L. Burton; Mark J. Solomon

The anaphase-promoting complex (APC) is a ubiquitin ligase responsible for promoting the degradation of many cell cycle regulators. One of the activators and substrate-binding proteins for the APC is Cdc20. It has been shown previously that Cdc20 can promote its own degradation by the APC in normal cycling cells mainly through a cis-degradation mode (i.e. via an intramolecular mechanism). However, how Cdc20 is degraded during the spindle assembly checkpoint (SAC) is still not fully clear. In this study, we used a dual-Cdc20 system to investigate this issue and found that the cis-degradation mode is also the major pathway responsible for Cdc20 degradation during the SAC. In addition, we found that there is an inverse relationship between APCCdc20 activity and the transcriptional activity of the CDC20 promoter, which likely occurs through feedback regulation by APCCdc20 substrates, such as the cyclins Clb2 and Clb5. These findings contribute to our understanding of how the inhibition of APCCdc20 activity and enhanced Cdc20 degradation are required for proper spindle checkpoint arrest.


Methods in Enzymology | 1995

[12] Expression, purification, and functional assay of Mss4

Janet L. Burton; Pietro De Camilli

Publisher Summary This chapter describes the expression of recombinant mammalian suppressor of Sec4 (Mss4) protein using the T7 RNA polymerase system and partial purification of the protein using gel filtration chromatography. This chapter also describes the filter-binding assay that has been used to demonstrate the GDP-releasing activity of Mss4. Mss4 was identified in a genetic screen as a suppressor of the temperature-sensitive yeast secretory mutant sec4-8 .The sec4-8 strain contains a point mutation in the Rab GTPase Sec4, which leads to the accumulation of post-Golgi secretory vesicles and growth arrest when these cells are shifted to the restrictive temperature of 37 o C. Rat brain cDNAs were expressed in the sec4-8 strain and analyzed for the property of restoring growth at the restrictive temperature. A direct interaction between Mss4 and the small GTPases has been demonstrated by in vitro studies in which recombinant Mss4 has been shown to promote GDP release from the wild-type Sec4 protein as well as with other selected Rib GTPases using a filter-binding assay.

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Elizabeth Garcia

Mansfield University of Pennsylvania

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