Podcasts about tom7

The BOB & TOM Show – June 30, 20266:00 Hour 6:00 – White Trash Noise Machine6:06 – Tom never watched Cops6:09 – Kristi and Pat disagree6:14 – John Cena discussion6:25 – Gad about Gaddis6:31 – "I could parallel park a train." – Josh6:35 – Letter: Listener has an Indianapolis 500 book signed by Marty Allen ("Hello dere")6:35 – Letter: "Thank you, Tom Griswold—I never thought I'd say that."6:50 – Concert floor incident and copyright discussion6:53 – Letter: Pick up a camera for the Brownie6:53 – Letter: Wallet found at a soccer game; crowd chants owner's name 7:06 – Tom switched iced teas and is feeling tired and grumpy7:07 – Jeff in studio7:09 – Dirty Ditties7:24 – Josh and Jeff compare chest hair7:25 – Concert floor incident was not an accident7:27 – Why aren't there more women's restrooms at concert venues?7:28 – Women using urinals7:29 – Tom says a "fridge cigarette" is a Diet Coke7:32 – Letter: Watching an old Columbo episode reminded a listener of Hugh Jackman and Tom7:37 – John Cena hair transplant discussion7:39 – Jeff compliments John Cena7:48 – Sports7:52 – Women's tennis players grunting discussion7:54 – Peter Tork discussion7:55 – Stephen Stills, the Monkees, and Charles Manson 8:03 – Josh jokes about Kristi having a crush on Charles Manson8:05 – First indoor fishing pond in a Moscow mall8:10 – Oldest living parrot discussion8:12 – Is a beak the same as a pecker?8:16 – "Not a grower or a shower—just there." – Jeff8:27 – Letter: Listener loved Jeff's hot dog trick8:27 – Josh invites everyone to run through his sprinkler8:30 – Discussion of P-Shot procedures8:34 – "We are at the dawn of Grandma Griswold." – Josh8:46 – Today in History 9:06 – Josh discusses his bladder scope procedure9:10 – Tom recalls having a bladder scope as a young man9:23 – Gen Z "Day Cap" day drinking trend9:25 – Michigan bill to make lemonade stands easier9:29 – Metallica donates $25,000 to a food bank9:30 – Jacket designed to harvest water from the air9:32 – Jeff says cottage cheese makes a terrible grilled cheese sandwich9:45 – Josh and Pat's trip to the grocery store9:50 – "Making Some Changes" – Josh 7:00 Hour8:00 Hour9:00 Hour Learn more about your ad choices. Visit podcastchoices.com/adchoices

The BOB & TOM Show – June 25, 2026 6:00 Hour6:08 Renting a movie theater is not bad financially – Tom6:12 Bunk bed talk6:16 Lean discussion6:25 Is it possible for you to speak without offending anyone, Tom? – Chick6:27 Pat's new T-shirt6:29 Letter: Things Kristi hates; Tom in general; marriage lasting6:32 BoBo Brazil discussion; Kristi dislikes him6:34 Josh tells someone to go play a penny whistle6:35 Letter: Name of kid from yesterday's letter and photo6:38 Letter: Listener gets excited about new dress socks, just like Tom6:41 Ace's car sounds like a lifeboat – Tom6:50 Letter: Watched Wild Bill Curry; not really in a cage6:53 Letter: Did you get the Christmas gift wrapped on Tom's desk Monday?6:54 Don't want anyone touching my stuff – Tom6:54 Letter: Stole a bag of ice like my hero Chick6:55 Letter: Rode with an elevator repairman in an elevator 7:00 Hour7:11 Tom's joke that no one got7:12 Chick got his girlfriend stolen by a camera guy7:14 Kristi found the perfect brownie with sea salt7:25 Store selling a box of ice for $4; ice cubes, not bags7:26 Tom hates crushed ice and curved ice cubes7:26 Explaining curved ice cubes – Tom7:31 Tom hates the movie Cocktail7:33 Sports7:47 Artist making custom cowboy hats for World Cup fans7:50 Tom has a hat wall7:53 My winter cowboy hat is made of beaver – Tom7:54 Woman at Kristi's grocery store wears Spock ears 8:00 Hour8:04 Chick discovers the Nextdoor app8:08 Cigarette butts in Tom's street; his dogs are eating them8:09 World record: Largest ballpoint pen, 21 feet 7 inches, created by an Iraqi man8:16 Ballpoint pens have to have a clicker8:30 Josh knocks the salt off his pretzels8:30 Soap in the anus discussion8:36 1,000 people invited to Taylor Swift's wedding8:38 Lean discussion8:49 Jess in studio8:49 Today in History8:52 You fill a room with people then get mad if they talk, Tom – Josh8:57 Tom dancing 9:00 Hour9:07 Al Jackson joins via Zoom9:09 Boarding planes – Al9:10 Tom not happy with jetways9:25 Missing giraffe in Texas9:33 Tom, could you eat a meal on the toilet while expelling? – Chick9:34 Bag stopper discussion – Al9:35 “Oh, calm down everyone” – Tom9:51 Couple celebrates 80th wedding anniversary, the Oak Anniversary Learn more about your ad choices. Visit podcastchoices.com/adchoices

The BOB & TOM Show – June 22, 2026 6:00 AM6:00 Tom lives by a code song – Pat6:03 Jeff in, Tom out6:06 Where is Cape Verde?6:07 Father's Day talk6:22 Letter: Betty plays tambourine in The Archies6:24 Letter: Keytar guy playing it on stage6:25 Letter: Looking for my vitamins, found them in my medicine glove box6:28 Letter: Playing dolls camping – “Daddy, could you hand me the fire sandwich?”6:28 Letter: Water seasoning = Crystal Light6:30 Put together a Little Tikes toy – took me 8 hours – Chick6:31 Letter: Had a spam dog – was great6:32 Letter: Was at a World Cup game – tickets were $1406:33 Tom has done a double decker?6:34 Letter: Melissa Etheridge = alternative person6:34 Letter: Sturgeon generals = group of sturgeons6:48 Sports6:53 Kristi hates feet6:55 I tend my feet once a month – Jeff6:55 Josh can feel flies on his head since he's bald 7:00 AM7:05 Kristi had her lazy eye fixed7:06 Pickleball fight7:07 Naked Pickleball song – Pat7:10 Man covered himself in baked beans; longest time in baked beans was 4 days7:14 Three Men and a Baby was horrible – Josh7:27 Sitting at Tom's desk, found an unwrapped Christmas gift – Jeff7:28 Let's re-wrap a Christmas gift for Tom7:28 Exercise makes people more generous – Kristi7:32 Pays for a gym membership, doesn't know which gym it is – Chick7:37 Kissed a guy – Josh7:38 Three guys peed their pants on purpose at a party – Josh/Chick7:47 Gave his cats artificial seafood – Josh7:50 A.I. robot plays table tennis7:53 FIFA athletes amazed by U.S. stores and food 8:00 AM8:04 In studio – Jess8:07 Betty Rubble vitamin was missing for 30 years8:11 125 MPH street racing can result in 85 years8:26 Police robot not doing his job8:27 People like snacking on bugs8:35 Used condoms, dead cat, maggots, boogers under seat – gross things found in a car on a date8:46 Today in History 9:00 AM9:04 Dad taught ballet – Pat Godwin9:24 3-D model for women's anatomy9:27 Letter: My dad turns 99 today, still works at a home improvement store and still drives9:28 Kristi: “I'm more lesbian than you?”9:36 All Minions are men; women not that stupid – creator comment9:47 Something that should be better than it is: Blondie – Josh Learn more about your ad choices. Visit podcastchoices.com/adchoices

The BOB & TOM ShowJune 17, 2026 6:00 – Walking to the liquor store – Pat Godwin6:06 – Had a Sting-Ray bike; got stolen – Chick6:09 – Letter: Death tree; need to find 8 names6:15 – Then Came Bronson theme discussion6:24 – Salt rifle6:25 – People eat flies6:25 – Letter: Husband bought a salt gun for our barn6:29 – Letter: Tom GTO fan; Mark Lindsay did a GTO commercial6:31 – Josh and Tom discussion6:33 – Letter: Josh mentions Tennessee Ernie Ford; Instagram was listening6:34 – “16 Tons” discussion6:36 – “Why do I think you guys think I'm mentally impaired?” – Chick6:49 – “Mule Train” discussion6:51 – Singing chickens6:53 – Letter: For reunion, have a task6:54 – Letter: 10-year class reunion invitation; responded with “Deceased” 7:04 – Far Side chicken stuck-in-concrete joke – Josh7:07 – Tom applauds sheep joke by Josh7:09 – Eye doctor discussion7:10 – My mom's hospital needed a bigger morgue – Tom7:12 – Letter: Donald Duck sneeze impression request for Pat7:24 – Chick's reunion job is contacting people7:25 – Letter: Mom, age 92, in charge of high school reunion; only six left7:27 – “Flirty Gertie,” world's oldest chicken song7:30 – ZZ Top song discussion7:32 – Foxborough7:33 – Sports7:35 – Drunkula7:49 – New York Knicks baby boomer coming soon7:52 – Jalen Rose original name discussion7:54 – Jalen Rose song7:56 – Knickers discussion 8:04 – More knickers discussion8:08 – SWR: Largest riding penny-farthing, 9 feet tall8:11 – Tom explains paved walking trails8:22 – Self-pleasure before bed may help sleep8:24 – Josh has a journal tracking self-pleasure8:28 – Poll: 30% have confidence in their sexual skills8:31 – Someone used a public pool as a bathroom8:44 – Today in History8:46 – Book about the Statue of Liberty's feet – Tom 9:03 – In studio: Jessica9:03 – Fried apple pie returns to McDonald's9:05 – Words that sound fake but are real: Bumfuzzle (confused)9:06 – Snollygoster9:06 – Friendlily9:07 – Pronk9:09 – Crapulent9:10 – Fartlek9:20 – Zoom with Alli Breen9:22 – Letter: Boyfriend does not want to get married; new job requires moving; should I go?9:27 – It's sweating season; men should shower regularly9:29 – Letter: Girlfriend talks about her ex all the time; I'm not jealous9:31 – Letter: Boyfriend keeps air conditioning extremely cold and won't turn it up9:35 – Letter: After an eight-year relationship, I slept with another guy9:45 – More words that sound like they shouldn't be real9:55 – Pilot wasn't a real pilot Learn more about your ad choices. Visit podcastchoices.com/adchoices

BOB AND TOM SHOW WEDNESDAY JUNE 3 20266:01 Attack of the wiener man-Here Come the Mummies6:05 Pat had shoulder surgery-He's fine6:10 Male members pick sides6:10 I'm a grower not a shower-Josh6:14 SIMPLI SAFE6:25 Letter-Brush my teeth in the shower6:28 Josh got in to puss laties class6:29 Letter-Husband loves my back scratches6:31 Big Bad John-Jimmy Dean/copyright6:33 Letter-Love Cajun Queen song by Jimmy Dean/copyright6:37 Small Sad Sam/copyright6:39 LEAN6:50 Cajun Queen Song explained by Tom6:53 Chicks Uber rating6:55 Little Bitty big John/copyright-Jimmy Dean7:06 Letter-look at my leg elbow=Knee-3 year old7:08 Letter-New Bob and Tom show-the Rotisserie man7:11 Letter-Remember they won't sell you a handgun if you are crying7:12 Devo song/copyright7:14 AMERICAN FINANCING7:23 Sports7:25 Have you ever wore spurs Tom?-Josh7:26 Had a cowboy kit as a kid-Tom7:33 Sex Mummy7:47 In Studio-Jess7:48 SWR-parade on Route 66 largest parade of classic cars-3596 cars7:53 Route 66-Nat King cole/copyright7:53 Tom says “before my time”7:55 Got your foreskin blown off in Civil War-Kristi8:03 Classic cars we would like to have8:10 Did anything happen to you after 1965, Tom?-Chick8:11 Vaginal health tips-mansplaining8:13 AURA FRAMES8:26 Windshield washing stations are empty at gas stations-Josh8:28 We need more flights-Josh8:29 More vaginal health tips-sleep naked8:30 I will never not sleep naked-Josh8:31 Josh's neighbor brought him chicken-he was in his boxers8:33 Vagina is a self -cleaning oven-Jess8:36 Alli is at the French open-Kristi calls her a bitch8:36 HYUNDAI8:47 Tom talks about his towel clips as a kid 8:48 Windows lead to the outside-Tom 8:50 Today in History 9:05 Dating a friends ex  9:07 Kissing Cousings Clip  9:08 Woman spent $50,000 to marry herself 9:13 SIMPLISAFE 9:26 More people charge their phones than put on sunscreen 9:26 Back to Tom's "Cowboy Kit"  9:31 Chick and Josh love tan lines 9:32 Woman suing Outback steak house-slipped on mashed potatoes 9:35 Please do not say my name in a non-comedy joke-Josh 9:37 AMERICAN FINANCING 9:49 Jess will be helping Pat play his guitar 9:51 Tom interviewing a DoDo bird 9:53 There is a McChicken on the menu Learn more about your ad choices. Visit podcastchoices.com/adchoices

The BOB & TOM Show — April 27, 2026 6:00 Go Out Drinking — Pat Daily6:05 Chick's Wordle streak ends today6:06 Letter: Listening to old shows; landlady with a nose ring at 80 years old6:09 Kristi would not dye her hair6:24 Chick brought Tom a tube of Fresh Balls6:28 Tom prefers hardwood6:31 Letter: Chernobyl meltdown anniversary (April 26)6:35 Story about a woman being robbed — Tom6:48 Letter: Original Jetsons now streaming6:49 Letter: No land snakes in Hawaii; kahula pork discussion6:51 Letter: Neighbor may have adopted peacocks on their farm6:52 “I Want Sugar” — Pat Godwin 7:03 Letter: April 25 was Ella Fitzgerald's birthday; also Tom's sister's birthday7:05 Song parody about Ella Fitzgerald — Pat Godwin7:05 Letter: Logan Stankoven — Tom's favorite hockey player7:06 Letter: Grandfather guarded tobacco storage7:08 Discussion: Smoking laws in London, England7:09 Letter: Love for ham radio7:22 Letter: SOS first used during the Titanic sinking7:22 “SOS” — ABBA7:24 “Fox on the Run” — Sweet7:25 “Ballroom Blitz” — Sweet7:36 Snack Kingdom: 6,000 snacks from around the world in China7:36 Unusual gum discussion — Tom7:49 Tom got married — congratulations to Tom and Kelly7:52 Tom wore Kelly's underwear (backwards)7:54 Chick comments on Tom's delicate fingers 8:06 Tom got married in the Bahamas; “Bahama Mama” comment8:07 Tom said “underpants” — Chick8:09 Discussion: Gen Z preference for tattoo engagement and wedding rings8:12 Josh has a Jarrod tattoo from Subway8:12 Josh says he is considered a “ruiner”8:13 “Butt hats” compared to tramp stamps — Tom8:25 Zoom interview: Greg Warren8:26 Bob soup talk clip8:27 Josh discusses being a cart corral worker8:27 Appalachian Trail discussion — Greg8:28 Trail takes 4–5 months to complete — Greg8:31 Trail nicknames8:33 Ten murders have occurred on the trail — Greg8:34 Copperhead snakes on the trail8:53 “Grave Wide Open” — song 9:03 Letter: Listener hiked the trail in six months; nickname was “Butt Spike”9:05 Story: Woman trapped after toilet collapse in Australia9:07 Toy Story 5 movie announcement9:21 Zoom interview: Jimmy Pardo9:22 Jimmy wearing a decoupage sweatshirt9:25 Jimmy shares kind words about Bob9:26 Jimmy's bumper sticker: “I Hate Jimmy Pardo”9:34 Helen Keller trading card discussion9:50 Trading card discussion continues Learn more about your ad choices. Visit podcastchoices.com/adchoices

Questions? Comments?Don and Tom question a surprising Wall Street Journal column arguing that annuities should become the default option in 401(k) plans. They explore why the idea is gaining traction, where the logic breaks down, and how the insurance industry benefits when complexity outpaces understanding. Along the way, they dig into the real shortcomings of annuities—fees, opacity, inflation risk, liquidity traps—and why “guarantees” often mask the true cost. Listener questions follow, covering tax-efficient stock cleanup at Schwab, spouse disagreements over individual stock picking, automatic ETF withdrawals at Vanguard, and building Dimensional portfolios inside Aspire plans.0:04 Don's rant: “What the world needs now is… more annuities?”1:20 WSJ's argument: make annuities the 401(k) default2:05 Why income complexity doesn't justify default annuities3:01 Do annuities actually solve longevity risk?3:29 Inflation, joint-life costs, and who really wins4:20 Insurance industry reputation and the unanswered criticisms5:15 High fees, opacity, and why mistrust is earned5:59 Are annuity sales tactics the real barrier?7:02 Should annuities be in 401(k)s at all? Don vs. Tom7:36 Why annuities are mostly sold, not bought9:10 Liquidity traps and major-life-event risks10:01 Why “plans” matter more than “products”10:57 Listener questions: why nobody calls anymore11:14 Q1: Selling a brokerage full of individual stocks at Schwab12:46 Q1b: How to convince a spouse who loves stock picking14:21 Indexing vs. anecdotal evidence16:21 SPIVA data and why active managers lose17:02 Q2: Can Vanguard automate ETF withdrawals?19:05 Fractional shares and why purchases are allowed20:25 Q3: Aspire 403(b) options and DFA overload23:46 How many DFA funds do you really need?24:44 Micro-cap risks and portfolio sprawl25:42 Tom's pumpkin-patch grandkid cameoLearn more about your ad choices. Visit megaphone.fm/adchoices

Alexander Repenning created AgentSheets, an environment to help kids develop computational thinking skills. It wrapped an unusual computational model with an even more unusual user interface. The result was divisive. It inspired so many other projects, whilst being rejected at every turn and failing to catch on the way Scratch later did. So in 2017, Repenning published this obit of a paper, Moving Beyond Syntax: Lessons from 20 Years of Blocks Programming in AgentSheets, which covers his findings over the years as AgentSheets evolved and transformed, and gives perspective on block-based programming, programming-by-example, agents / rule / rewrite systems, automata, and more. This is probably the most "normal" episode we've done in a while — we stay close to the text and un-clam many a thought-tickling pearl. I'm saying that sincerely now to throw you off our scent the next time we get totally lost in the weeds. I hear a clock ticking. Links $ Do you want to move beyond syntax? Frustrated by a lack of syntactic, semantic, or pragmatic support? Join our Patreon! Choose the tier that best reflects your personal vision of the future of coding. Get (frequently unhinged) monthly bonus content. Most of all: let us know that you enjoy this thing we do, and help us keep doing it for years to come. Argos, for our non-UK listeners. They were acquired by future TodePond sponsor, Sainsbury's. Once again, I am asking for your Marcel Goethals makes a lot of cool weird stuff and is a choice follow. Scratch isn't baby programming. Also, you should try this bizarre game Ivan programmed in 3 blocks of Scratch. Sandspiel Studio is a delightful block-based sand programming simulator automata environment. Here's a video of Lu and Max introducing it. Simple Made Easy, a seminal talk by Rich Hickey. Still hits, all these years later. Someday we'll do an episode on speech acts. Rewrite rules are one example of rewriting in computing. Lu's talk —and I quote— "at Cellpond", was actually at SPLASH, about Cellpond, and it's a good talk, about —and I quote— "actually, what if they didn't give up on rewrite rules at this point in history and what if they went further?" Oh yeah — Cellpond is cool. Here's a video showing you how it works. And here's a video studying how that video works. And here's a secret third thingthat bends into a half-dimension. Here's Repenning's "rule-bending" paper: Bending the Rules: Steps Toward Semantically Enriched Graphical Rewrite Rules I don't need to link to SimCity, right? You all know SimCity? Will Wright is, arguably, the #1 name in simulation games. Well, you might not have caught the fantastic article Model Metropolis that unpacks the (inadvertently?) libertarian ideology embodied within the design of its systems. I'd also be remiss not to link to Polygon's video (and the corresponding write-up), which lend a little more colour to the history. Couldn't find a good link to Blox Pascal, which appears in the paper Towards "Second Generation" Interactive, Graphical Programming Environments by Ephraim P. Glinert, which I also couldn't find a good link to. Projectional / Structural Editor. Here's a good one. Baba is You Vernacular Programmers Filling Typed Holes with Live GUIs is, AFAIK, the most current canonical reference for livelits. I'm not linking to Minecraft. But I will link to the Repeater 32 Checkboxes Wiremod is a… you know what, just watch this. Chomsky Hierarchy The Witness Ivan wrote a colorful Mastodon thread surveying the history of the Connection Machine. Harder Drive is a must-watch video by the inimitable Tom7. Also couldn't find a good link for TORTIS. :/ Programming by Example (PbE) Buffalo buffalo Buffalo buffalo buffalo buffalo Buffalo buffalo Alex Warth, one of the most lovely humans Ivan knows, is a real champion of "this, because that". Ivan's magnetic field simulations — Magnets! How do they work? Amit Patel's Red Blob Games, fantastic (fantastic!) explorable explanations that help you study various algorithms and techniques used in game development. Collaborative diffusion — "This article has multiple issues." Shaun Lebron, who you might know as the creator of Parinfer, made a game that interactively teaches you how the ghost AI works in Pac-Man. It's fun! Maxwell's Equations — specifically Gauss's law, which states that magnetic fields are solenoidal, meaning they have zero divergence at all points. University of Colorado Boulder has a collection of simulations called PhET. They're… mid, at least when compared to building your own simulation. For instance. Music featured in this episode: snot bubbles ! Send us email, share your ideas in the Slack, and catch us at these normal places: Ivan: Mastodon • Website Jimmy: Mastodon • Website Lu: Login • Website See you in the future! https://futureofcoding.org/episodes/073Support us on Patreon: https://www.patreon.com/futureofcodingSee omnystudio.com/listener for privacy information.

In this episode, Dave and Jamison answer these questions: I am working at my first job as a software engineer for 2 1/2 years now. I really enjoy working as a programmer and I'm super excited about the tech industry in general. However, sometimes I feel like I'm too excited about everything. I spent a lot of time reading blog posts, watching tutorials or taking online courses. I think about what books to read and what languages to learn all the time. Not everything but a big part of it happens during my working hours. While I know that “loving to learn” in general is considered a positive trait, I feel like I might take it a bit too far and I should focus more on the actual tasks I have - especially, because I think my coworkers spend much less time keeping up to date with everything. What is a reasonable amount of time to spent on these things during working hours and beyond? How do I know I spend too much time not working on my actual tasks? How can I make sure I learn the right things that are useful to my career? Love the show and wish you the best. Thanks for your advice! I landed a new job that nearly tripled my salary realative to the job I'm about to leave (yes, I was horribly underpaid)! The stories and tips from this podcast really helped me out but I also landed this job through Hired.com (the podcast sponsor). Any good tips regarding leaving a job when you know your boss will be furious that you're leaving? Also, should I tell my boss which company I'm going to when he asks (he definitely will)? Show Notes Tom7, the most amazing YouTuber of all time: https://www.youtube.com/c/suckerpinch https://www.youtube.com/watch?v=qTBAW-Eh0tM - anagraphs

Eric Potter is writing code to play NES games. This episode is sponsored by Smartsheet. Show Notes: Nintaco R.O.B. video from the NES Works video series Tom Murphy aka Tom7 Check out the NES AI videos from Tom7. I reference part 2 in this podcast. Check out starting at 18:11 especially. Podcast: Retronauts Book: Racing the Beam Aptera Some of the games mentioned in this episode: Castlevania Bomberman Super Mario Brothers Tecmo Super Bowl Heavy Barrel Skate or Die NintacoProxy on NuGet ROMhacking.net which includes memory locations, translations, and more. Book: I Am Error Event: PonCon, Sep 22nd 2018 Eric Potter is on Twitter. Want to be on the next episode? You can! All you need is the willingness to talk about something technical. Music is by Joe Ferg, check out more music on JoeFerg.com!

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into the organelle. The translocase is a multi-subunit complex composed of seven subunits: two receptor proteins, Tom70 and Tom20, and five components which form the core complex, Tom40, Tom22, Tom7, Tom6, and Tom5. In this thesis it is shown that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane but not for its assembly into the TOM complex. Structural characteristics of Mim1 required for its function were studied in detail. Mim1 forms homooligomeric structures via its transmembrane segment which contains two helix-dimerization GXXXG/A motifs. The homooligomerization is a precondition for the function of Mim1 in mediating the integration of Tom20 into the mitochondrial outer membrane.

Die Translokase der mitochondrialen Außenmembran TOM-Komplex erkennt alle in Mitochondrien zu importierenden Proteine und vermittelt den Transfer in oder über die Membran. Der Mechanismus der Translokation ist allerdings bisher nur teilweise verstanden. Strukturelle Informationen über den TOM-Komplex können Fragen nach den Detailschritten beantworten helfen. Aus dem Schimmelpilz Neurospora crassa wurde der TOM-Komplex so aufgereinigt, dass Ausbeute und Reinheit strukturelle Untersuchungen mittels Proteinkristallographie ermöglichten. Kristalle des TOM-Komplexes wurden gewonnen und Beugungsexperimente durchgeführt. Die Auflösung der erhaltenen Kristalle des TOM-Komplexes war nicht ausreichend, um Aussagen über die Raumgruppe oder den strukturellen Aufbau des Komplexes treffen zu können. Deshalb wurde das Reinigungsverfahren weiter optimiert sowie eine Reihe von Mutanten des TOM-Komplexes untersucht. Bisher konnte jedoch keine Verbesserung der Beugungsdaten erreicht werden. Zu Beginn dieser Arbeit waren vier Untereinheiten des TOM-Core-Komplexes von N. crassa bekannt: Tom40, Tom22, Tom7 und Tom6. Im ansonsten vergleichbar aufgebauten TOM-Core-Komplex von S. cerevisiae war noch eine weitere Komponente Tom5 beschrieben worden. Daher wurde im Vorfeld der Kristallisationsexperimente die Zusammensetzung des N. crassa TOM-Komplexes massenspektrometrisch analysiert, wobei Tom5 als eine weitere Tom-Untereinheit in N. crassa identifiziert werden konnte. Aufgrund von Experimenten mit S. cerevisiae wurde eine Rolle des Tom5 im Proteinimport in Mitochondrien postuliert. Im Gegensatz hierzu hatte Tom5 in N. crassa keinen Einfluß auf den Proteinimport. Auch auf die Assemblierung des TOM-Komplexes und das Wachstum der Zellen wirkte sich eine Deletion von Tom5 in N. crassa nicht negativ aus. Die Ergebnisse dieser Arbeit lassen allerdings eine solche Funktion auch in Hefe als fraglich erscheinen. Vielmehr deuten die hier vorgelegten Befunde auf eine strukturelle Funktion von Tom5 bei der Stabilisierung des TOM-Komplexes hin. Um weitere neue Komponenten des Import- und Assemblierungsapparates der mitochondrialen Außenmembran zu finden, wurde eine massenspektrometrische Analyse der Proteine von isolierten Außenmembranvesikeln aus N. crassa durchgeführt. Hierbei wurde Mim1 als bisher unbekanntes Außenmembranprotein identifiziert. Mim1 liegt in einem 300 kDa-Komplex vor und es wurde eine essentielle Funktion von Mim1 bei der Assemblierung des TOM-Komplexes nachgewiesen.

The TOM complex, a multisubunit assembly in the mitochondrial outer membrane, mediates targeting and membrane translocation of virtually all nuclear-encoded mitochondrial preproteins analyzed so far. In the present study the mechanisms by which the TOM complex recognizes different precursor proteins and translocates them across the outer membrane were investigated. In a first part of study the isolated TOM complex was analyzed for its ability to interact with preproteins with N-terminal targeting signals. The TOM translocase was found to bind precursor proteins efficiently in a specific manner in the absence of chaperones and lipids in a bilayer structure. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was mediated also by protease-treated TOM holo complex that contains almost exclusively Tom40. The TOM core complex consisting of Tom40, Tom22, Tom5, Tom6 and Tom7 represents a molecular machine that can recognize and partially translocate mitochondrial precursor proteins. In a second part of study the interaction of BCS1 precursor with the TOM complex was investigated. BCS1 belongs to the group of proteins with internal, non-cleavable import signals. The information for import and intramitochondrial sorting of BCS1 was localized to the region consisting of amino acid residues 1-126. Three sequence elements were identified in this region: (i) a transmembrane domain (amino acid residues 45-68), (ii) a presequence-like helix (residues 69-83), and (iii) an import-auxiliary sequence (residues 84-126). The contribution of each of these elements to import was studied. The transmembrane domain was found not to be required for stable binding to the TOM complex. The Tom receptors (Tom70, Tom22 and Tom20), as determined by peptide scan analysis, had no affinity for peptides corresponding to the transmembrane domain. They did interact with the presequence-like helix, yet the highest binding was to the region covering residues 92-126. This latter region represents a novel type of signal with targeting and sorting function. It is recognized by all three known mitochondrial import receptors demonstrating their capacity to decode various targeting signals. The results of the present study suggest that the BCS1 precursor crosses the TOM complex as a loop structure. This is in contrast to preproteins with cleavable presequences which enter the TOM complex in a linear fashion with the N-terminal first. Once the precursor emerges from the TOM complex, all three structural elements are essential for the intramitochondrial sorting to the inner membrane.

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.

Die Biogenese von Mitochondrien erfordert den Eintransport cytosolisch synthetisierter Vorstufenproteine über die mitochondriale Außenmembran. Der TOM-Komplex in der Außenmembran erkennt die in die Mitochondrien zu importierenden Vorstufenproteine, bindet sie, ermöglicht den Transfer von zumindest Teilen davon über die mitochondriale Außenmembran und die Integration von Membranproteinen in die Außenmembran. Auf der Grundlage bestehender Untersuchungen des TOM-Holo-Komplexes wurden in dieser Arbeit verschiedene Subkomplexe des TOM-Komplexes aus Neurospora crassa isoliert, biochemisch und biophysikalisch charakterisiert. Zudem wurde eine neue Komponente des TOM-Komplexes identifiziert: Tom5, eine kleine Komponente von etwa 5 kDa mit Sequenzhomologie zu Tom5 von Saccharomces cerevisiae. Der in dieser Arbeit isolierte TOM-Core-Komplex besteht aus den Protein-untereinheiten Tom40, Tom22, Tom7, Tom6 und Tom5; gegenüber dem TOM-Holo-Komplex fehlen ihm die Rezeptorkomponenten Tom70 und Tom20. Der TOM-Core-Komplex weist eine Molekülmasse von ca. 400 kDa und eine Stöchimetrie der Komponenten Tom40 : Tom22 : Tom7 : Tom6 von 8 : 4 : 2 : 1-2 auf. Er kann in vitro präsequenzabhängig bis zu 8 Vorstufen-proteine pro Komplex binden. Elektronenmikroskopische Bilder des TOM-Core- Komplexes zeigen eine symmetrische Doppelringstruktur mit zwei durchgehenden Poren von etwa 2,1 nm Durchmesser. Der TOM-Core-Komplex bildet in Übereinstimmung damit Kanäle mit zwei Leitfähigkeits-niveaus, die zwei Poren entsprechen. Die Bevorzugung von Kationen und die Eigenschaft, durch mitochondriale, positiv geladene Präpeptide selektiv und spezifisch inhibiert zu werden, belegen die Rolle des TOM-Core-Komplexes bei der Proteintranslokation. TOM-Core-Komplex, dessen hydrophile Domänen von Tom22 und den kleinen Toms durch limitierte Proteolyse weitgehend abgedaut wurden, zeigte in den durchgeführten Untersuchungen nahezu identische Binde-, Kanal- und Struktureigenschaften wie der unbehandelte Core-Komplex. Die Grundstruktur der Proteintranslokase der mitochondrialen Außenmembran Zusammenfassung - 132 - kann somit hinreichend durch Tom40 und die membrandurchspannenden Domänen von Tom22, Tom7, Tom6 und Tom5 stabil gebildet werden. Weiterführende Experimente mit isoliertem Tom40 bestätigten dies. So bildet isoliertes Tom40 oligomere Strukturen mit einer mittleren Molekülmasse von ca. 350 kDa. Tom40 zeigte sich in Transmissions-EM-Bildern überwiegend als Einlochpartikel. In Übereinstimmung hiermit weisen die vom Tom40- Komplex gebildeten Kanäle eine Leitfähigkeit von nur der Hälfte der Leitfähigkeit des TOM-Core-Komplexes mit zwei Poren auf. Ein kleiner Teil des isolierten Tom40 bildet Zweilochpartikel. Tom40 ist also in der Lage, die Grundstruktur des TOM-Komplexes zu bilden, wie sie für den TOM-Core-Komplex gefunden wurde. Infrarot- und Circulardichroismus-Spektren von isoliertem Tom40 führen zu dem Schluß, daß ein einzelnes Tom40-Protomer keinen Kanal mit β -Barrel-Struktur bilden kann, sondern daß dazu mehrere Tom40 zusammenwirken müssen.