Quantitative imaging of TATA-binding protein in living yeast cells

Patterson, George H.; Schroeder, Stephanie; Bai, Yu; Weil, P A; Piston, David W.

doi:10.1002/(sici)1097-0061(19980630)14:9<813::aid-yea280>3.0.co;2-2

Cited by 18 publications

(6 citation statements)

References 33 publications

(50 reference statements)

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“…Hence an active process limits the residency times, thereby reducing them from what is expected based on in vitro binding constants. Consistent with this notion, results indicate that TBP is limiting for transcription in both yeast (38) and mammalian cells (39,40). At first glance a surprising feature of our results is that TBP FRAP dynamics in live yeast cells (Fig.…”

Section: Resultssupporting

confidence: 89%

Regulation of TATA-binding protein dynamics in living yeast cells

Sprouse

Karpova

Mueller

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Although pathways for assembly of RNA polymerase (Pol) II transcription preinitiation complexes (PICs) have been well established in vitro, relatively little is known about the dynamic behavior of Pol II general transcription factors in vivo. In vitro, a subset of Pol II factors facilitates reinitiation by remaining very stably bound to the promoter. This behavior contrasts markedly with the highly dynamic behavior of RNA Pol I transcription complexes in vivo, which undergo cycles of disassembly/reassembly at the promoter for each round of transcription. To determine whether the dynamic behavior of the Pol II machinery in vivo is fundamentally different from that of Pol I and whether the static behavior of Pol II factors in vitro fully recapitulates their behavior in vivo, we used fluorescence recovery after photobleaching (FRAP). Surprisingly, we found that all or nearly all of the TATA-binding protein (TBP) population is highly mobile in vivo, displaying FRAP recovery rates of <15 s. These high rates require the activity of the TBP-associated factor Mot1, suggesting that TBP/chromatin interactions are destabilized by active cellular processes. Furthermore, the distinguishable FRAP behavior of TBP and TBP-associated factor 1 indicates that there are populations of these molecules that are independent of one another. The distinct FRAP behavior of most Pol II factors that we tested suggests that transcription complexes assemble via stochastic multistep pathways. Our data indicate that active Pol II PICs can be much more dynamic than previously considered.fluorescence recovery after photobleaching ͉ Mot1 ͉ TFIID T ranscription preinitiation complex (PIC) formation involves the assembly of general transcription factors (GTFs) at appropriately remodeled chromatin templates. Regulation of RNA polymerase (Pol) II transcription initiation can occur by many different mechanisms involving facilitated recruitment of PIC components or stimulation of their activities at the promoter. Although the diversity of such regulatory mechanisms is widely appreciated, comparatively little is known about the dynamic behavior of GTFs in vivo vis a vis their interactions with each other and with chromatin. Most nuclear proteins studied to date, including a number of gene-specific transcription factors and chromatin-modifying enzymes, display highly dynamic behavior in vivo (1-3). In many cases, this dynamic behavior is energy-independent (4). Even chromatin structural proteins once thought to be stably bound to DNA display dynamic behavior in vivo (3-9). However, the dynamic behavior of GTFs is largely unexplored. In vitro, a PIC scaffold nucleated by TATA binding protein (TBP) is very stable, a property that facilitates multiple rounds of transcription at an activated promoter (10). A stable TBP association with chromatin is supported by analysis in human cells, which yields a slow fluorescence recovery time for TBP of 20 min (11). In striking contrast, in vivo analysis of RNA Pol I PICs yields a much more dynamic picture, with fluores...

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Section: Resultssupporting

confidence: 89%

Regulation of TATA-binding protein dynamics in living yeast cells

Sprouse

Karpova

Mueller

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…This was also the case for a chromatin-associated protein, a GFP fusion of TBP (Patterson et al, 1998) (our unpublished results). To learn whether hypertonic shock per se is responsible for the relocation of Fpr3p and Nop1p, we have also used 0.5 M KCl and 1 M sorbitol.…”

Section: (Our Unpublished Results)supporting

confidence: 69%

“…E-mail address: amt10@po.cwru.edu. Wente, 1998]), or pGFP-TBP (GFP-TBP, HIS3, CEN [Patterson et al, 1998]) by standard procedures and maintained on appropriate selective plates. DL376 transformants carrying pDL468 (GAL-PKC1 wt -HA, URA3, CEN [Gray et al, 1997]), pDL469 (GAL-PKC1p K853R -HA, URA3, CEN [Gray et al, 1997]), pBM743 (GAL-PKC1p R398A , URA3, CEN [Delley and Hall, 1999]), pHPS29 (PKC1 C434S, C437S, C514S, C517S , TRP1, CEN [Jacoby et al, 1997]), or pHPS30 (pPKC1 wt , TRP1, CEN [Jacoby et al, 1997]) were maintained on selective plates containing 0.5 M sorbitol.…”

Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

Perturbation of the Nucleus: A Novel Hog1p-independent, Pkc1p-dependent Consequence of Hypertonic Shock in Yeast

Nanduri

Tartakoff

2001

MBoC

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Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade. In contrast, protein kinase C (Pkc1p) and the "cell integrity" MAP kinase cascade are critical for the response to hypotonic shock. We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm. We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects. In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not. Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins. Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously. The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock. INTRODUCTIONThe cellular effects of changes in tonicity have been investigated extensively (Robbins et al., 1970;Banuett, 1998;Gustin et al., 1998;Lang et al., 1998). In particular, analysis of the yeast Saccharomyces cerevisiae has elucidated major roles for two MAP kinase signaling pathways: the Pkc1p "cell integrity" pathway, which is stimulated by hypotonic shock (Watanabe et al., 1994;Davenport et al., 1995), and the Hog1p pathway, which is stimulated by hypertonic shock (Brewster et al., 1993;Posas and Saito, 1997). Each pathway is required for long-term survival in the corresponding medium. Hypertonic shock profoundly affects the actin cytoskeleton (Chowdhury et al., 1992;Mulholland et al., 1994;Delley and Hall, 1999).The Pkc1p pathway can receive input from plasma membrane glycoproteins of the Wsc family and from the Rho GTPases and their regulatory factors, for example in the context of control of cell polarization and cell wall biosynthesis (Gustin et al., 1998). Interestingly, some signaling events that require Pkc1p appear not to require the corresponding downstream MAP kinase cascade, which is linked to activation of the transcription factors Rlm1p and Swi4p/ Swi6p (Lee and Levin, 1992;Gustin et al., 1998;Delley and Hall, 1999; Li et al., 2000). In contrast, the Hog1p pathway is initiated by a two-component phosphorelay involving the cell surface transmembrane protein Sln1p and the cytosolic protein Ypd1p (Posas et al., 1996). Stimulation of this pathway causes phosphorylation and transient nuclear entry of the terminal kinase, Hog1p, as well as of the transcription factor Msn2p (Ferrigno et al., 1998;Gö rner et al., 1998;Reiser et al., 1999). The downstream substrate(s) of Hog1p kinase is not known.We have observed recently that arrest of the secretory pathway in yeast inhibits nuclear import and causes many nucleolar and nucleoplasmic proteins to relocate to the cytoplasm. These events (the "arrest of secretion response") a...

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“…These huntingtin constructs containing 78 glutamine repeats and tagged with either YFP or CFP (httQ78-YFP, httQ78-CFP) or 23 glutamine repeats (httQ23-YFP) were used to examine interactions with the polyglutamine aggregate-associated proteins, TBP (TBP-YFP), CBP (YFP-CBP), and Hsp70 (Hsp70-YFP). These chimera proteins are functional as previously described (14,(42)(43)(44)(45). Co-expression of httQ78-YFP and httQ78-CFP resulted in the appearance of aggregates with both proteins uniformly distributed throughout (Fig.…”

Section: Detection Of "Ring" Structures Formed By Co-expression Of Tbmentioning

confidence: 95%

Huntingtin and Mutant SOD1 Form Aggregate Structures with Distinct Molecular Properties in Human Cells

Matsumoto¹,

Kim²,

Morimoto

2006

Journal of Biological Chemistry

105

116

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Expression of many proteins associated with neurodegenerative disease results in the appearance of misfolded species that readily adopt alternate folded states. In vivo, these appear as punctated subcellular structures typically referred to as aggregates or inclusion bodies. Whereas groupings of these distinct proteins into a common morphological class have been useful conceptually, there is some suggestion that aggregates are not homogeneous and can exhibit a range of biological properties. In this study, we use dynamic imaging analysis of living cells to compare the aggregation and growth properties of mutant huntingtin with polyglutamine expansions or mutant SOD1 (G85R/G93A) to examine the formation of aggregate structures and interactions with other cellular proteins. Using a dual conditional expression system for sequential expression of fluorescence-tagged proteins, we show that mutant huntingtin forms multiple intracellular cytoplasmic and nuclear structures composed of a dense core inaccessible to nascent polypeptides surrounded by a surface that stably sequesters certain transcription factors and interacts transiently with molecular chaperones. In contrast, mutant SOD1 (G85R/G93A) forms a distinct aggregate structure that is porous, through which nascent proteins diffuse. These results reveal that protein aggregates do not correspond to a single common class of subcellular structures, and rather that there may be a wide range of aggregate structures, perhaps each corresponding to the specific disease-associated protein with distinct consequences on the biochemical state of the cell.Accumulation of abnormal protein deposits as aggregates or inclusion bodies is a common cytological feature of a number of disease states as represented by clinically related neurodegenerative diseases. The mutant proteins that initiate protein aggregates in many of these diseases have been identified: A␤ in Alzheimer disease, PrP in prion diseases, ␣-synuclein in Parkinson disease, huntingtin in Huntington disease, tau in tauopathy, and SOD1 in familial amyotrophic lateral sclerosis (1-3). Although these proteins do not share distinctive common features in their respective primary sequences, they have all been shown to adopt alternate conformational states and form misfolded protein structures that appear visually as aggregates and inclusion bodies that correlate with disease pathology. There is increasing evidence to support a "toxic gain-of-function" mechanism by which misfolded protein and aggregate structures lead to a dominant pathological phenotype (2, 4).A molecular basis for proteotoxicity is the aberrant interactions between aggregation-prone proteins and other cellular proteins. This is in part supported by in vivo polyglutamine disease models in which aggregates have been shown to contain specific transcription factors, cytoskeletal, autophagy, and degradative proteins as well as molecular chaperones (5-14). The recruitment and sequestration of these cellular proteins has been proposed to lead to functional deplet...

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Quantitative imaging of TATA-binding protein in living yeast cells

Cited by 18 publications

References 33 publications

Regulation of TATA-binding protein dynamics in living yeast cells

Regulation of TATA-binding protein dynamics in living yeast cells

Perturbation of the Nucleus: A Novel Hog1p-independent, Pkc1p-dependent Consequence of Hypertonic Shock in Yeast

Huntingtin and Mutant SOD1 Form Aggregate Structures with Distinct Molecular Properties in Human Cells

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