Quick academic help
Don't let the stress of school get you down! Have your essay written by a professional writer before the deadline arrives.
General pathway of chitin biosynthesis in biological systems.
(vi) The CHS3 gene, as well as all the genes responsible for its intracellular movement and localization, is required for both stress- and GlcN-induced chitin synthesis. We find we must revise the previous indication that the targeting gene, CHS6, is not required for stress-related chitin synthesis (). Although there is indeed some bypassing of the chs6 mutation in the chs6 fks1 double mutant, this is reflected as an increase in chitin levels of less than 20% of the chitin made in wild-type cells. Interestingly, the chs6 mutation is more prone to a bypass when combined with gas1 mutation. And finally, when the chs5 deletion is combined with gas1 deletion, the bypass is exemplified by restoration of chitin levels to those of wild-type cells.
To further explore the role of the precursor pool in chitin synthesis by Chs3p, the levels of UDP-GlcNAc in several cell wall mutants were quantitated. Soluble components were extracted from the cytosol of logarithmically growing cells with formic acid. Extracted metabolites were incubated with alkaline phosphatase to remove phosphomonoester intermediates. This treatment reduces the complexity of the high-pressure liquid chromatographyprofile, since diester-linked metabolites are limited to nucleotide sugars and a few coenzymes. Figure shows the levels of UDP-GlcNAc in cells grown in complete medium alone and with added GlcN. In each case the level was measured three to five times, and the values were consistent. Surprisingly, similar levels of UDP-GlcNAc were also found in cultures grown to saturation overnight.
Chitin synthases in yeast and fungi.
Chs3p synthesizes about 90% of the chitin in S. cerevisiae. Levels of Chs3p are virtually unaltered during the yeast life cycle (). However, temporal changes in its subcellular location result from being secreted to and endocytosed from the plasma membrane. Chs3p transits through the endoplasmic reticulum/Golgi secretory pathway to the plasma membrane early in the formation of a daughter cell. Once the daughter cell is full size, Chs3p is retrieved by endocytosis into “chitosomes,” intracellular vesicles related to the trans-Golgi network and early endosomes (, ). Several proteins—Chs4p (Skt5p), Chs5p, Chs6p, and Chs7p—regulate Chs3p enzymatic activity and trafficking through the endoplasmic reticulum to the plasma membrane (reviewed in references and ). Loss of any of these proteins results in a reduction of the chitin concentrationin the cell wall to levels comparable to those observed when Chs3p itself is absent. Chs4p serves a dual role of binding with Chs3p to form an active complex and localizing the active complex to the bud-neck region by also binding to the septin ring through Bni4p (). The resulting synthesis of a ring of chitin reinforces the bud-neck region during cell division. For vegetatively grown cells, the amount of Chs4p is limiting and therefore impacts chitin synthesis by its availability to complex with Chs3p (). Chs7p is involved specifically in the exit of Chs3p from the endoplasmic reticulum (). Chs5p and Chs6p have been identified as components required for transport of secretory and/or endocytic vesicles to the plasma membrane (, , ). The clathrin AP-1 complex has recently been shown to also be important in the process of retrieval of Chs3p by endocytosis and its recycling into the secretory pathway ().
In this paper we report our recent findings on the factors that contribute to the regulation of chitin synthesis. We studied a number of single and double mutants, which elevate or decrease chitin levels, and examined the effect on chitin levels of addition of GlcN or α-factor to the growth medium. We show here that there is a direct correlation between Gfa1p activity, the pool of metabolic intermediates, and chitin synthesis. Finally, since the increase in chitin levels associated with treatment of wild-type cells with GlcN is similar to the increase in chitin levels associated with the cell wall stress response, we investigated the relevant whole-genome transcription responses.
Chitin synthases in yeast and fungi
Another obvious question was whether treatment of cells with GlcN would lead to transcription of any of the genes found to be “turned on” in strains with mutations affecting cell wall structure, since in both cases there is a major increase in the synthesis of lateral cell wall chitin. Also, we wanted to know whether GlcN simply produces—either directly or indirectly—a cell wall stress. In that case, the transcription profile following addition of GlcN would be similar to that seen, for example, in the fks1 mutant. We designed two experiments. In one, the cells were grown first in YPD medium and then for 1 to 2 h after supplementation with GlcN (to 15 mM). In the other, cells were grown in the presence of GlcN overnight, diluted in fresh medium containing 15 mM GlcN, and again grown to mid-log phase (these are referred to below as cells exposed to “steady-state” conditions). Analysis of genomewide expression was carried out in triplicate in all cases, and the average intensities of the mRNA signals showing a significant change are reported in Table .
An obvious question is whether chitin synthesis is proportional to, or is in any way “driven” by, the cellular UDP-GlcNAc level. The data show that this is not the case. Although we and Lagorce et al. () found that chitin synthesis is proportional to the cellular level of Gfa1p, the enzyme responsible for the formation of GlcN-6-P, it is clear that UDP-GlcNAc concentrations are increased to only moderately higher levels in mutant strains that are making more than twice as much chitin as wild-type cells. More dramatically, when chitin synthesis is driven not by Gfa1p but by added GlcN, the level of UDP-GlcNAc is actually lowest in cells that show the highest rate of synthesis. It is thus clear that the UDP-GlcNAc concentration alone does not control the rate of chitin formation. In summary, since chitin synthesis is proportional to Gfa1p but not to UDP-GlcNAc concentrations, it is likely that either Gfa1p itself or another GlcN metabolite plays a role in the activation and/or localization of Chs3p. However, the recent results of Valdivia et al. () show that control of Chs3p targeting may be complex and subject to secondary mutations. These investigators demonstrated that mutations in proteins of the clathrin AP-1 complex allow extensive bypassing of the Chs6p requirement for lateral wall chitin synthesis.
Why choose our assistance?
As soon as we have completed your work, it will be proofread and given a thorough scan for plagiarism.
Our clients' personal information is kept confidential, so rest assured that no one will find out about our cooperation.
We write everything from scratch. You'll be sure to receive a plagiarism-free paper every time you place an order.
We will complete your paper on time, giving you total peace of mind with every assignment you entrust us with.
Want something changed in your paper? Request as many revisions as you want until you're completely satisfied with the outcome.
We're always here to help you solve any possible issue. Feel free to give us a call or write a message in chat.
Synthesis and characterization of polypyrrole grafted chitin
GlcN is presumed to enter cells by way of hexose transport systems and to be phosphorylated by hexokinase(s). The resulting GlcN-6-P is a normal intermediate in the chitin synthesis pathway, and the increased levels of metabolic intermediates must initiate the increased chitin formation observed. GlcNAc, on the other hand, either is not transported into the cell or is not phosphorylated.
Insect Chitin Biosynthesis and Inhibition - USDA
The increase in chitin synthesis that occurs in response to GFA1 overexpression is probably not a response to cell wall stress. It may represent a bypass of the stress response, i.e., stress may normally lead first to stimulation of GFA1, which in turn stimulates chitin synthesis directly or indirectly (see Discussion for an analysis of this hypothesis). If this is the case, then increasing the GlcN-6-P pool by simply adding GlcN to the growth medium might also lead to increased chitin synthesis. It has been shown that GlcN can readily be taken up and phosphorylated by S. cerevisiae (). Typical results are shown in Fig. . The chitin content of cells treated with GlcN (0 to 23 mM in YPD medium) increases from 4 to 5 nmol of GlcNAc per mg (wet weight) to about 14 nmol. GlcN concentrations higher than 23 mM in the medium have a toxic effect on cells. We also measured the rate of chitin synthesis in yeast cells exposed to 15 mM GlcN over time (Fig. ). There was no apparent lag in chitin synthesis, as chitin levels nearly doubled in the first hour. After 4 h, chitin content approached its new steady-state level, as found for cells grown overnight with GlcN. Upon examination by fluorescence microscopy using Calcofluor to stain chitin, large budded cells as well as mother cells showed increased fluorescence in their lateral walls following culture in 15 mM GlcN (data not shown).
chitin biosynthetic process | SGD
Other genes were virtually unaffected after 2 h of GlcN treatment. The largest functional group of transcripts up-regulated in the steady state represents genes involved in mating, sporulation, and cell cycle arrest. Activation of these genes in the steady state may be a secondary effect of GlcN and an adaptive response to new growth conditions. In the gene transcription pattern there is essentially no overlap (with the exception of IME1, ATF1, YDL241W, SRD1, YNL129W, and PLB3) between the cell wall stress response in fks1Δ strains and exposure to GlcN (Fig. ), although in these two cases chitin synthesis is stimulated to about the same extent. Since most of the overlapping genes are involved in mating and sporulation, this may again be an adaptation to steady-state exposure to GlcN rather than a direct impact of GlcN on their transcription.
How it works
You submit your order instructions
We assign an appropriate expert
The expert takes care of your task
We send it to you upon completion
Average quality score
"I have always been impressed by the quick turnaround and your thoroughness. Easily the most professional essay writing service on the web."
"Your assistance and the first class service is much appreciated. My essay reads so well and without your help I'm sure I would have been marked down again on grammar and syntax."
"Thanks again for your excellent work with my assignments. No doubts you're true experts at what you do and very approachable."
"Very professional, cheap and friendly service. Thanks for writing two important essays for me, I wouldn't have written it myself because of the tight deadline."
"Thanks for your cautious eye, attention to detail and overall superb service. Thanks to you, now I am confident that I can submit my term paper on time."
"Thank you for the GREAT work you have done. Just wanted to tell that I'm very happy with my essay and will get back with more assignments soon."