The “Pro” Sequence of the Sporulation-Specific ς Transcription Factor ςE Directs It to the Mother Cell Side of the Sporulation Septum

1999 ◽  
Vol 181 (19) ◽  
pp. 6171-6175 ◽  
Author(s):  
Jingliang Ju ◽  
W. G. Haldenwang
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Fluorescent Protein ◽  
Amino Terminus ◽  
Cell Compartment ◽  
Intracellular Location ◽  
Specific Protease ◽  
Green Fluorescent

ABSTRACT ςE, a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-ςE). Activation of ςE occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-ςE amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-ςE and green fluorescent protein (GFP) to visualize the intracellular location of pro-ςE by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-ςE::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-ςE::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-ςE::GFP, and presumably pro-ςE, to the mother cell is likely to be the reason why ςE activity is restricted to that compartment.

scholarly journals Tethering of the Bacillus subtilis σE Proprotein to the Cell Membrane Is Necessary for Its Processing but Insufficient for Its Stabilization

2003 ◽  
Vol 185 (19) ◽  
pp. 5897-5900 ◽  
Author(s):  
Jingliang Ju ◽  
W. G. Haldenwang
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Cell Membrane ◽  
Cytoplasmic Membrane ◽  
Fluorescent Protein ◽  
Amino Terminus ◽  
Membrane Association ◽  
Developmentally Regulated ◽  
Sufficient Cause

ABSTRACT σE, a sporulation-specific transcription factor of Bacillus subtilis, is synthesized as an inactive proprotein with a 27-amino acid extension at its amino terminus. This “pro” sequence is removed by a developmentally regulated protease, but when present, it blocks σE activity, tethers σE to the bacterium's cytoplasmic membrane, and promotes σE stability. To investigate whether pro-σE processing and/or stabilization are tied to membrane sequestration, we used fluorescent protein fusions to examine the membrane binding of SigE variants. The results are consistent with membrane association as a prerequisite for pro-σE processing but not as a sufficient cause for the proprotein's stability.

scholarly journals A Four-Dimensional View of Assembly of a Morphogenetic Protein during Sporulation in Bacillus subtilis

1999 ◽  
Vol 181 (3) ◽  
pp. 781-790 ◽  
Author(s):  
Kirsten D. Price ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Outer Surface ◽  
Mother Cell ◽  
Fluorescent Protein ◽  
Mature Spore ◽  
Time Lapse ◽  
Homologous Protein ◽  
C Terminus ◽  
Morphogenetic Protein ◽  
Green Fluorescent

ABSTRACT We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacteriumBacillus subtilis. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium Clostridium acetobutylicum, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, likespoIVA, are under the control of the mother cell transcription factor ςE. One such gene wasspoVM, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.

scholarly journals Polar Localization and Compartmentalization of ClpP Proteases during Growth and Sporulation in Bacillus subtilis

2008 ◽  
Vol 190 (20) ◽  
pp. 6749-6757 ◽  
Author(s):  
James Kain ◽  
Gina G. He ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Mother Cell ◽  
Regulatory Networks ◽  
Fluorescent Protein ◽  
The Other ◽  
Spatial Control ◽  
Polar Localization ◽  
Cell Compartments ◽  
Green Fluorescent ◽  
Necessary And Sufficient

ABSTRACT Spatial control of proteolysis is emerging as a common feature of regulatory networks in bacteria. In the spore-forming bacterium Bacillus subtilis, the peptidase ClpP can associate with any of three ATPases: ClpC, ClpE, and ClpX. Here, we report that ClpCP, ClpEP, and ClpXP localize in foci often near the poles of growing cells and that ClpP and the ATPase are each capable of polar localization independently of the other component. A region of ClpC containing an AAA domain was necessary and sufficient for polar localization. We also report that ClpCP and ClpXP proteases differentially localize to the forespore and mother cell compartments of the sporangium during spore formation. Moreover, model substrates for each protease created by appending recognition sequences for ClpCP or ClpXP to the green fluorescent protein were preferentially eliminated from the forespore or the mother cell, respectively. Biased accumulation of ClpCP in the forespore may contribute to the cell-specific activation of the transcription factor σF by preferential ClpCP-dependent degradation of the anti-σF factor SpoIIAB.

scholarly journals Substrate Requirements for Regulated Intramembrane Proteolysis of Bacillus subtilis Pro-σK

2005 ◽  
Vol 187 (3) ◽  
pp. 961-971 ◽  
Author(s):  
Heather Prince ◽  
Ruanbao Zhou ◽  
Lee Kroos
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Terminal Segment ◽  
Intramembrane Proteolysis ◽  
Regulated Intramembrane Proteolysis ◽  
N Terminus ◽  
Green Fluorescent ◽  
A Charge

ABSTRACT During sporulation of Bacillus subtilis, pro-σK is activated by regulated intramembrane proteolysis (RIP) in response to a signal from the forespore. RIP of pro-σK removes its prosequence (amino acids 1 to 20), releasing σK from the outer forespore membrane into the mother cell cytoplasm, in a reaction catalyzed by SpoIVFB, a metalloprotease in the S2P family of intramembrane-cleaving proteases. The requirements for pro-σK to serve as a substrate for RIP were investigated by producing C-terminally truncated pro-σK fused at different points to the green fluorescent protein (GFP) or hexahistidine in sporulating B. subtilis or in Escherichia coli engineered to coexpress SpoIVFB. Nearly half of pro-σK (amino acids 1 to 117), including part of sigma factor region 2.4, was required for RIP of pro-σK-GFP chimeras in sporulating B. subtilis. Likewise, pro-σK-hexahistidine chimeras demonstrated that the N-terminal 117 amino acids of pro-σK are sufficient for RIP, although the N-terminal 126 amino acids, which includes all of region 2.4, allowed much better accumulation of the chimeric protein in sporulating B. subtilis and more efficient processing by SpoIVFB in E. coli. In contrast to the requirements for RIP, a much smaller N-terminal segment (amino acids 1 to 27) was sufficient for membrane localization of a pro-σK-GFP chimera. Addition or deletion of five amino acids near the N terminus allowed accurate processing of pro-σK, ruling out a mechanism in which SpoIVFB measures the distance from the N terminus to the cleavage site. A charge reversal at position 13 (substituting glutamate for lysine) reduced accumulation of pro-σK and prevented detectable RIP by SpoIVFB. These results elucidate substrate requirements for RIP of pro-σK by SpoIVFB and may have implications for substrate recognition by other S2P family members.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

scholarly journals Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

2001 ◽  
Vol 12 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
Jack Rohrer ◽  
Rosalind Kornfeld
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Lysosomal Hydrolases ◽  
Intracellular Location ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by “uncovering” enzyme (UCE), which removes a coveringN-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: 488YHPL and C-terminal 511NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

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