Podcasts about dhfr

In this episode, we dive into the critical process of methylation and its significance in detoxification, mood regulation, and overall cellular function. We explain the step-by-step conversion of dietary folate to its active form and how gene mutations can impair this process, leading to reduced SAMe levels, high homocysteine, and various health issues. Finally, we discuss practical strategies to support methylation through dietary choices and targeted supplementation. Topics: 1. Introduction - Overview of previous discussions on Lyme and its coinfections (Babesia and Bartonella) - Transition to the importance of supporting detoxification pathways 2. The Role of Methylation in Health - Definition and significance of methylation - Importance in detoxification, mood, energy, and gene activation 3. Methylation Process - Biochemical explanation of methylation - Key molecules involved: S-adenosylmethionine (SAMe), methionine, ATP - Dietary sources and conversion process of folate (vitamin B9) 4. Synthesis of S-adenosylmethionine (SAMe) - Step-by-step process from dietary folate to SAMe - Enzymatic conversions: DHFR, MTHFR - Role of active folate (L-5-MTHF) in methylation 5. Gene Mutations and Their Impact - Common MTHFR gene mutations: C677T and A1298C - Effects of reduced MTHFR enzyme activity - Consequences: elevated homocysteine levels, reduced SAMe production 6. Consequences of Impaired Methylation - DNA methylation and gene expression - Neurotransmitter synthesis and mental health - Detoxification efficiency - Accumulation of unmetabolized folic acid 7. Supporting Methylation - Dietary strategies: - Emphasizing natural folate intake - Food sources: leafy greens, legumes, fruits - Supplementation strategies: - Ensuring adequate intake of vitamin B12 and B6 - Using L-5-MTHF instead of synthetic folic acid - Trimethylglycine (TMG) supplementation Thank you to our episode sponsor: GenuinePurity Use code CHLOE20 to get $20 off GenuinePurity NMN GenuinePurity NR GenuinePurity Spermidine GenuinePurity Trans-Resveratrol GenuinePurity Fisetin Thanks for tuning in! Get Chloe's Book Today! "⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠75 Gut-Healing Strategies & Biohacks⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠" If you liked this episode, please leave a rating and review or share it to your stories over on Instagram. If you tag @synthesisofwellness, Chloe would love to personally thank you for listening! Follow Chloe on Instagram ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠@synthesisofwellness⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠ Follow Chloe on TikTok @chloe_c_porter Visit ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠synthesisofwellness.com⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠ to purchase products, subscribe to our mailing list, and more! Or visit ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠linktr.ee/synthesisofwellness⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠ to see all of Chloe's links, schedule a BioPhotonic Scanner consult with Chloe, or support the show! --- Support this podcast: https://podcasters.spotify.com/pod/show/chloe-porter6/support

Question: What cofactors are needed to synthesize and recycle BH4? Short Answer: Zinc, magnesium, potassium, and niacin are the cofactors needed for the synthesis and recycling of BH4. Folate and methylation are not involved, though high-dose folate or folic acid could hypothetically hurt BH4 recycling since both are recycled by dihydrofolate reductase (DHFR). This is a clip from a live Q&A session open to CMJ Masterpass members. In addition to this episode, you can access two other free samples using this link: https://chrismasterjohnphd.substack.com/p/questions-on-vitamin-d-sulfate-synthesis In that batch of free episodes you will also find the answers to these questions: Is It Important to Get Vitamin D Sulfate Specifically From the Sun? How Much Iron Can We Absorb At Once? If you want to become a Masterpass member so you can participate in the next live Q&A, or so you can have access to the complete recording and transcript of each Q&A session, you can save 10% off the subscription price for as long as you remain a member by using this link to sign up: https://chrismasterjohnphd.substack.com/qanda Learn more about the Masterpass here: https://chrismasterjohnphd.substack.com/about This snippet is from the February 15, 2023 AMA. The full recording and transcript is reserved for Masterpass members. Here is a preview of what's included: Is It Important to Get Vitamin D Sulfate Specifically From the Sun? How Much Iron Can We Absorb At Once? What nutrients are important for long-term PPI use? For how long does transferrin saturation respond to recent iron-rich food? Muscle spasms: creatine, creatinine, sodium, and potassium. Hematologists ignore iron saturation. How to detox arsenic? Could folic acid supplements impair BH4 recycling? How to increase butyrate? More on hematologists and transferrin saturation. Here's a link to the full AMA: https://chrismasterjohnphd.substack.com/p/recording-and-transcript-of-the-february  Access the show notes, transcript, and comments here.

Have you ever looked at the nutritional recommendations for pregnancy and wondered how accurate they are? In this deep dive mini episode, we compare Folic acid and Folate as it relates to recommended health guidelines. Topics covered in this episode: Folate vs Folic Acid Genetics and gene variants DHFR, MTHFR Methylation Bio-individual nutrient needs Genetic capacity for enzyme production Foods highest in Folate How to shop for a prenatal vitamin Processed foods and the origin of Folic AcidChris Kresser Podcast Episode #179:Nutrients Dr. Ben Lynch Dirty GenesFollow @healthinstitute on Instagram! instagram.com/healthinstitute Join The Health Institute Newsletter! www.thehealthinstitute.com/wellness-weekly

Question: Does folic acid act differently in the body than natural folate? | Masterjohn Q&A Files #68They don't really. Everything that is said bad about folic acid is sort of true to an extent but has been completely exaggerated in some circles. What happens is you have an enzyme called dihydrofolate reductase, or DHFR. Its purpose is not to metabolize synthetic folic acid obviously because that folic acid molecule doesn't exist in the food supply. Its normal purpose is that every time that you use folate to participate in processes outside of methylation, such as DNA synthesis, you wind up producing dihydrofolate as a byproduct. DHFR recycles that and turns it into tetrahydrofolate, or THF. Tetrahydrofolate is what has the methyl group added to make methylfolate. The question is, does that synthetic folic acid, we call that unmetabolized folic acid, does that cause harm? There are scientific hypotheses that it might, and it might, but there's no conclusive evidence of that. That's one side of the argument against synthetic folic acid. The other side of the argument is now that you are giving the DHFR enzyme more work, that means that might be detracting from the work that it has in recycling dihydrofolate that came out of the DNA synthesis reactions to make tetrahydrofolate. People think that they just cut out white flour and therefore they're better off. No. You cut out white flour, now you need to do more work to make sure that you are actually getting your nutrients from whole foods because if you were eating six pieces of white toast that you didn't have to worry about getting nutrients from whole foods and now you do. This Q&A can also be found as part of a much longer episode, here: https://chrismasterjohnphd.com/podcast/2019/03/30/ask-anything-nutrition-march-4-2019 If you would like to be part of the next live Ask Me Anything About Nutrition, sign up for the CMJ Masterpass, which includes access to these live Zoom sessions, premium features on all my content, and hundreds of dollars of exclusive discounts. You can sign up with a 10% lifetime discount here: https://chrismasterjohnphd.com/q&a

Book: Dirty Genes http://amzn.to/2nHlhBf www.drbenlynch.com Dirty Genes  http://learntruehealth.com/dirty-genes/ Dirty genes is a significant factor why we do not reach an optimal level of health. Our body needs healthy genes to function properly. But unfortunately, many people are still unaware of this vital aspect of health.  That’s why we’re so lucky to have Dr. Ben Lynch here with us today to explain everything you need to know about dirty genes.  Childhood Interest   Dr. Ben Lynch was 16 years old when he was first introduced to biochemistry in high school. He thought it was the best class he ever had because it taught him about how the body works.    Because Dr. Ben Lynch became fascinated with how our body was wired, he went on to pursue a medical degree in college. But it was not long before he realized that standard medicine and the conventional route was not for him.   The turning point in his career path was when Dr. Ben Lynch got sick during a trip to India. Finding a cure for his illness, he was introduced to a peculiar form of medicine. Dr. Ben Lynch recalls that the one treating him checked his tongue, pulse, and gave a weird prescription. Surprisingly, he felt better after 40 minutes!    That experience eventually led Dr. Ben Lynch to pursue a career as a Naturopathic Doctor by getting his degree at Bastyr University. But Dr. Ben Lynch still felt it wasn’t enough until he developed an interest in Environmental Medicine. This led him to discover epigenetics.   Focus On Epigenetics   Dr. Ben Lynch was very much concerned about how a poor environment can make people very ill and develop dirty genes.  When he learned that genes were a significant factor in the entire process, he started to dive deeper into genetic research.   “Our genes require tools. Most of the tools come from the food that we eat. If we are providing our body with healthful, wholesome foods, our genes get the tools they can use to function. Such as protein, carbs, fats, vitamins, minerals, water, air, and sunlight,” said Dr. Ben Lynch.  He adds, “We give genes those tools through nutrition, breathing properly, hydration and avoid doing stuff that makes them do excessive work like chemicals in the environment, wi-fi signals, and polluted air.”   And that’s so true! I first heard about epigenetics from Dr. Joe Wallach. He wrote the book, Epigenetics: The Death of the Genetic Theory of Disease Transmission. The book explains how nutrition supports the genes to be healthy. It’s a very informative book, and I recommend all you listeners to include this in your reading list.     Air Purifiers   Pollutants in the environment are unavoidable. But because they cause dirty genes, Dr. Ben Lynch says one way to clear the air is using air purifiers. Air purifiers can clear the air of chemicals and elements like smoke, pollen, and dust.     Good brands of air purifiers include IQAir and Alen Air. But Dr. Ben Lynch puts in a word of caution if and when you decide to get one.   “Air purifiers have an engine.  I recommend plugging it in and turn on outside to let it off the gas,” advises Dr. Ben Lynch. “Because nowadays, everything is wrapped in plastic. When you turn it on, there are so many volatile organic compounds that are being emitted.”   Dr. Ben Lynch also advises avoiding having carpets if you want to avoid having dirty genes. Because apparently, carpets are sponges that hold dirt. There are also a lot of other odd compounds in carpets. It traps dust and humidity, attracts molds and dust mites.    Gas And Cooking   We also know that gas is a toxic substance.  That’s why Dr. Ben Lynch strictly advises that gas is something that needs to be outside. So, if you’re cooking, you need to be using your exhaust fan.    “Smoke from oils have formaldehyde, destroy genes and burdens the body. Then a lot of people are taking antacids, which are inhibiting their primary detoxification system,” said Dr. Ben Lynch. “People are also loading up with Tylenol. This depletes primary antioxidants. So be wary of what you buy over the counter.”   Dr. Ben Lynch says it is also bad to inhale smoke from cars. The carbon monoxide binds to the hemoglobin. And when carbon monoxide seeps into our tissues, it can eventually kill us.    “You increase cardiovascular disease risk from inhaling smoke. People who are exercising in more polluted areas are also increasing cardiovascular risk,” Dr. Ben Lynch said. “That has to do with a gene called NOS 3, which is an important gene to keep clean.”    Dirty Genes Course   To those who would like to know more about the causes of dirty genes, do sign up for Dr. Ben Lynch’s course. The course provides a lot of insights on how to optimize your eating habits, and to have a better experience without supplements.    “The course guides people through the fundamentals of life. It also educates people why we need folate to make our body function right,” said Dr. Ben Lynch.   According to Dr. Ben Lynch, folate is essential in things like:   regulating white blood cells  fighting infection  regulating red blood cells so we can breathe  regulate our platelets  nourish our neurotransmitters so we can think  helps repair DNA when chemicals invade into our system  essential in kids’ growth  makes skin healthy since our skin creates skin cells all the time   Importance Of Folic Acid   Folic acid is a form of folate and is one of the B vitamins that we need. The U.S. recommended daily allowance of folic acid is 400 micrograms. These may be gained through food or dietary supplements.   “Folic acid binds preferentially to the folate receptors and folate transporters in the body. It goes through the DHFR (dihydrofolate reductase) gene.  This gene can handle 200 micrograms of folic acid before it gets saturated,” said Dr. Ben Lynch.   But Dr. Ben Lynch says some people have a genetic problem with this particular gene. Apparently, the folic acid can’t get through the DHFR gene. It has to go somewhere, so it ends up staying in the blood. Researchers call it unmetabolized folic acid.   “What unmetabolized folic acid does, is that it will get to your folate transport protein. Folic acid blocks transportation of the folate and getting inside the cell,” explains Dr. Ben Lynch.    Dr. Ben Lynch also reveals that people can be functionally folate-deficient. This is because they are functionally depriving their cells of the number one type of folate in their body which is methylfolate. Common side effects include experiencing brain fog, moodiness, and an unexplainable toxic feeling.   StrateGene   Ready to dive deeper into how genes work? Dr. Ben Lynch developed a genetic report called StrateGene. It uses 23andMe instead of ancestry because according to Dr. Ben Lynch, ancestry mainly has ancestral DNA. In order words, he says you don’t have clinically relevant genes on www.ancestry.com to run through a genetic report.    StrateGene also provides you a map so you can see what dirties up the genes. It also provides a quick glance at how vitamins, minerals, medication, and chemicals interfere with certain things.   “If you see any condition run in your family, there is most likely a genetic component there. And if you lead a lifestyle that is healthy, the chances of you struggling with that condition is lower,” said Dr. Ben Lynch.   But Dr. Ben Lynch says the more important factor is finding out what genes you inherited that is probably causing your illness. Knowing which genes are born dirty can give you an insight on where you should be spending more time and caution. And the cool thing is that you can support yourself through very targeted actions that will help the weaker genes.    What are Dirty Genes?   Now many of you may be wondering what dirty genes are. Dr. Ben Lynch explains that dirty genes are genes that are not functioning at it’s best. One can inherit dirty genes from your parents. And it usually does not function as well as it should be in your particular environment.    “You can inherit it. Genes also get dirty by sitting in the garage after turning on your car or turning on your gas stove and not turning the hood on. Dirty genes are also from eating unhealthy or from chronic stress,” said Dr. Ben Lynch.  Juicing, Raw Foods And Cooking   Dr. Ben Lynch advises that if you cook your leafy green vegetables, steam them for a short period. That’s where you’ll get your folate. Apparently, the longer you cook, the more your folate will be destroyed.    “Leafy greens are great. But if you have a weak constitution and digestion, eating raw, cold vegetables can be hard on you. So you might want to steam them,” Dr. Ben Lynch advises. ” If you have a frail palate, steam veggies and have soup. Keep in mind your body temperature, the power of your digestion and how you feel after. Listen to your body.”   Dr. Ben Lynch claims to love juicing as well. He says he prefers using a Blendtec over Vitamix because it is not as noisy. I like Blendtec, too. But I have heard feedback from friends that it breaks down.    Incidentally, I had the Vitamix expert Lenny Gale on my show a couple of times.  His website Life is No Yoke is packed with info on the different Vitamix models as well as yummy recipes using Vitamix. So I invite everyone to check out his website.    Histamine Intolerance   Dr. Ben Lynch also recommends a good brand of probiotics to keep our microbiome healthy. Some health conditions like headaches, runny nose, asthma and leaky gut is a sign that one may have histamine intolerance.   Histamine in the stomach and small intestines is good because it stimulates the stomach acid, movement of stool and immune response. But it is not good to have too much histamine in our gut because it could lead to digestive issues like diarrhea, and acid reflux.   Link Between Vaccines And Genes   Dr. Ben Lynch shares that vaccines have a role in specific environments for certain people. But he believes vaccines are hurting our kids and pregnant women. The same goes for flu shots.    Ideally, Dr. Ben Lynch recommends that parents should wait until their child is at least 12 years old before getting them vaccinated instead of when they are newborns. This is because any heavy metal will interfere with a plethora of genes.    “You can take all the supplements you want, but if you have metals, genes won’t work well. Avoid metals like aluminum because they interfere with neurotransmitters in the brain,” said Dr. Ben Lynch.   He adds, “I am not pro-vaccine, but I don’t think they are all bad. I just think if we do use them, they should be used selectively.”   I recently had a great interview with Dr. Paul Thomas on vaccines, and the need to have a vaccine-friendly plan. We talked extensively about choosing when and which vaccines are safe so do check out that episode as well.   Dr. Benjamin Lynch, ND received his Cell and Molecular Biology, BS from the University of Washington and his doctorate in Naturopathic Medicine (ND) from Bastyr University. His passion for identifying the cause of disease-directed him towards nutrigenomics and methylation dysfunction.    Currently, Dr. Benjamin Lynch researches, writes and presents worldwide on the topic of MTHFR, methylation defects and genetic control. He is the President of www.SeekingHealth.com, a supplement company oriented towards disease prevention and health promotion. He also founded and directed www.seekinghealth.org, an educational institution providing specialized training for both health professionals and consumers.  He lives in Seattle, WA with his wife, Nadia, and three boys, Tasman, Mathew and Theodor.    Get Connected With Dr. Ben Lynch! Official Website   Seeking Health   Seeking Health.org   StrateGene   Facebook   Twitter   Instagram   Youtube    Book by Dr. Ben Lynch Dirty Genes     Recommended Reading by Dr. Ben Lynch The One Thing by Gary Keller     The Links You Are Looking For: Support Us on Patreon & Join the Learn True Health Book Club!!! 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Stop taking synthetic folic acid. Folic acid is poorly converted to reduced (active) folate. Folic acid also competes with biopterin for DHFR (dihydrofolate reductase). Biopterin is crucial for neurotransmitter production.

The vast majority of mitochondrial proteins are encoded in the nucleus and synthesized as precursor proteins on cytosolic ribosomes. After translation, these precursor proteins are imported in a largely, if not completely, unfolded state into one of the four mitochondrial subcompartments, the outer membrane, the intermembrane space, the inner membrane or the matrix. Once the precursor proteins reach their compartment of destination, they can fold into the functionally active three-dimensional native structure. Therefore, internal mitochondrial folding systems are needed in each subcompartment to assist folding of these precursor proteins upon import. Members of several “classical” chaperone families are present in the mitochondrial matrix and have been shown to support import and folding of newly imported polypeptides. However, folding of proteins in the mitochondrial intermembrane space is only poorly understood. Recently, a disulfide relay system in the intermembrane space that mediates import and folding was described, but this system is limited to proteins that form disulfide bonds. For the majority of intermembrane proteins, folding helpers that promote folding have not yet been discovered. In order to identify general folding helpers of the intermembrane space, the well studied model substrate mouse dihydrofolate reductase (DHFR) was targeted to the mitochondrial intermembrane space of S. cerevisiae and its folding analyzed. DHFR assumes its mature fold in the intermembrane space and heat shock induces DHFR aggregation. Interestingly, aggregation is counteracted by an ATP-dependent process. The i-AAA protease Yme1 that is anchored in the inner mitochondrial membrane and exposes its functional domains to the intermembrane space was able to prevent the aggregation of DHFR. A number of proteins of diverse structural and functional classes were found in the aggregate fractions of mitochondria lacking Yme1. Amongst them were factors that are involved in the establishment and maintenance of the mitochondrial ultrastructure, lipid metabolism, protein translocation and respiratory growth. Considering the diversity of the proteins affected in the absence of Yme1 and their function in mitochondria, the pleiotropic effects of the deletion of Yme1 can be readily explained. The findings of the present in vivo study confirm previous hints to a chaperone-like function of Yme1 resulting from in vitro experiments. Yme1 thus has a dual role as protease and as chaperone and occupies a key position in the protein quality control system of the mitochondrial intermembrane space.

Levels of drug resistance of Plasmodium falciparum strains against antimalarials have increased in Laos. In several studies, chloroquine (CQ) resistance has been associated with point mutations in the Pfcrt and pfmdr genes, and sulphadoxine/pyrimethamine (S/P) resistance with point mutations in the genes of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). We combined a study of these molecular markers with an in vivo antimalarial drug sensitivity study in Attapeu province in the south of Lao PDR. We treated 100 patients with either CQ, S/P or a combination of both. In the CQ group, Pfcrt mutations showed a very high sensitivity (100%) but a low specificity (12.5%) to predict resistance. The combination of mutations in the Pfcrt and pfmdr genes was highly specific and had a positive predictive value of 100%. Mutations in the DHPS gene showed a high correlation with the development of resistance. The prevalence of mutations in the DHFR gene, especially codon 108 Asn, was predictive with high sensitivity (100%) but low specificity. Isolates derived from patients treated with a combination of both drugs showed a high correlation between the mutation in codon 437 of DHPS gene and in vivo-resistance (odds ratio 16.00, CI). The study provides evidence for the existence of antimalarial drug resistance in the south of Lao PDR, and offers a molecular method to predict resistance.

Background: Malaria parasites that carry the DHFR-mutation I164L are not only highly resistant to sulfadoxine-pyrimethamine but also to the new antimalarial drug chlorproguanil-dapsone. The spread of this mutation in Africa would result in a public health disaster since there is a lack of effective alternatives that are both affordable and safe. Up to now, this mutation has only been described in Asian and Latin-American countries. The objective of this study was to assess the prevalence of this mutation in African isolates of Plasmodium falciparum that have been imported into Europe through travellers. Methods: TropNetEurop is a network for the surveillance of travel-associated diseases and seems to cover approximately 12% of all malaria cases imported into Europe. Within this network we screened 277 imported African isolates of P. falciparum with the help of PCR- and enzyme-digestion-methods for the antifolate-resistant mutation I164L. Results: The I164L mutation was not detected in any of the isolates tested. Discussion: Continuous molecular surveillance of mutations in P. falciparum, as it is practised within TropNetEurop, is an essential tool for the understanding and early detection of the spread of antimalarial drug resistance in Africa.

Die Proteintranslokase in der mitochondrialen Außenmembran (TOM-Komplex) ist verantwortlich für die Erkennung von mitochondrialen Präproteinen und deren Translokation über die mitochondriale Außenmembran. Das Ziel dieser Arbeit bestand in der Klonierung und Charakterisierung von bislang nicht identifizierten Komponenten des TOM-Komplexes in Neurospora crassa sowie in der Charakterisierung der Bindung von Präproteinen an den isolierten TOM-Komplex. Dabei wurden folgende Ergebnisse erzielt: Es wurden zwei bislang unbekannte, ca. 6 bzw. 7 kDa grosse Komponenten des Neurospora crassa TOM-Komplexes, Tom6 und Tom7, identifiziert. Deren Gene wurden mittels Durchmusterung einer cDNA-Phagenbibliothek sowie einer sortierten genomischen DNA-Bibliothek identifiziert und sequenziert. Das TOM6-Gen umfasst drei Exons und zwei Introns, während das TOM7-Gen vier Exons und drei Introns enthält. Die Aminosäuresequenzen von Neurospora crassa Tom6 und Tom7 weisen eine hohe Ähnlichkeit zu denen von Tom6 und Tom7 aus anderen Organismen auf. Dabei erstreckt sich der homologe Bereich bei Tom7 über die gesamte Aminosäuresequenz, während er bei Tom6 auf den carboxyterminalen Bereich beschränkt ist. Für beide Proteine wurde jeweils eine potentielle Transmembrandomäne an ihrem Carboxyterminus vorausgesagt. Sowohl Tom6, als auch Tom7 sind integrale Bestandteile des TOM-Core-Komplexes und befinden sich in engem Kontakt zu anderen Komponenten des TOM-Komplexes. Es konnte mit Hilfe von chemischen Quervernetzungsexperimenten gezeigt werden, daß sich Tom6 und Tom7 im TOM-Komplex von Neurospora crassa in direkter räumlicher Nähe zu Tom 40 befinden. Außerdem konnte ein direkter Kontakt zwischen Tom6 und Tom22 nachgewiesen werden, welcher durch Bindung des Präproteins pSu9-DHFR moduliert wird. Ein weiterer Schwerpunkt bei der Charakterisierung von Neurospora crassa Tom6 und Tom7 bestand in der Untersuchung des Imports dieser Proteine in Mitochondrien sowie deren Assemblierung in bereits bestehende TOM-Komplexe. Sowohl Tom6, als auch Tom7 konnten in vitro in Mitochondrien importiert werden und in bereits bestehende TOM-Komplexe assemblieren. Dabei benutzen sie teilweise den generellen Importweg von Präproteinen in Mitochondrien. Der Import von Tom6 umfasst zwei nicht miteinander gekoppelte Schritte. Zunächst findet eine vom Carboxyterminus vermittelte Interaktion mit Komponenten des TOM-Komplexes statt, es folgt die Assemblierung in den TOM-Komplex. Die Assemblierung von Tom6 in den TOM-Komplex setzt eine spezifische Interaktion des aminoterminal an die Transmembrandomäne angrenzenden Bereichs mit anderen TOM-Komponenten voraus. Daneben ist eine Interaktion der Transmembrandomäne von Tom6 mit dem aminoterminal an die Transmembrandomäne angrenzenden Bereich von Tom6 essentiell für die korrekte Assemblierung von Tom6 in den TOM-Komplex. Im Gegensatz zu anderen Außenmembranproteinen kommt bei Neurospora crassa Tom6 positiv geladenen Aminosäuren im an die Transmembrandomäne angrenzenden Bereich keine Bedeutung für den Import zu. Ein weiterer Aspekt der vorliegenden Arbeit bestand in der Untersuchung einiger Aspekte der Bindung des mit Fluoreszenzfarbstoff markierten Präproteins pSu9-DHFR an den isolierten TOM-Komplex unter Anwendung der Fluoreszenzkorrelationsspektroskopie. Die Bindung dieses Präproteins an den TOM-Komplex ist reversibel und wird spezifisch von der Präsequenz vermittelt. Die apparenten Bindungskonstanten betragen 1,3 nM für den TOM-Holokomplex sowie 3,4 nM für den TOM-Core-Komplex. Ein wichtiges Merkmal der Bindung von pSu9-DHFR an den TOM-Komplex sind elektrostatische Wechselwirkungen, da eine Erhöhung der Ionenstärke im Reaktionspuffer eine drastische Verminderung der Bindung zur Folge hatte. Des weiteren geht die Bindung von pSu9-DHFR an den TOM-Komplex einher mit der Entfaltung der DHFR. Eine Verhinderung der Entfaltung der DHFR durch Komplexierung mit Methotrexat führte zu einer stark verminderten Bindung von pSu9-DHFR an den TOM-Komplex.

Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through the contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 30 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes.

The precursor of F0-ATPase subunit 9 was bound to mitochondria in the absence of a mitochondrial membrane potential (delta psi). Binding was mediated by a protease-sensitive component on the mitochondrial surface. When delta psi was reestablished, bound precursor was directly imported without prior release from the mitochondrial membranes. A chimaeric protein consisting of the complete subunit 9 precursor fused to cytosolic dihydrofolate reductase (DHFR) was also specifically bound to mitochondria in the absence of delta psi. Two other fusion proteins, consisting either of the entire presequence of subunit 9 and DHFR or of part of the presequence and DHFR, were imported in the presence of delta psi. In the absence of delta psi, however, specific binding to mitochondria did not take place. We suggest that the hydrophobic mature part of subunit 9 is involved in the delta psi-independent binding of the subunit 9 precursor to receptor sites on the mitochondrial surface.