Autophagy: Exploring Cellular Recycling and Protein Degradation Mechanisms

[Music] probably you feel tired but let me start my talk okay I these days I always start

this kind of slide by everything Sciences our system of knowledge accumulated by human humanity as a whole

and science is a social activity so we cannot be independent of the air we live so today I will start my history I was

born in 1944 these five that are just half years before that World War 2 was over at that time everybody in Japan is

so pure and we have so many troubles about their food and and we have I had there's hard time my mother suffered

from TB for a long time and when I was in this high school I was interested in chemistry but so I want to be a chemist

when I entered the University of Tokyo but as you know 1960s there was a really good time for gesture establishing the

molecular biology so I interested in the molecular biology was pretty much so I started Wonder lab Khatami majalis lab

working on the ribosome a protein live some structure and the protein since its initiation mechanism so my career is

pretty much interested in the protein synthesis in within the cell and as you know biology neither's

just the organism to work I have been already working on this small tiny is single

so organism Saccharomyces LDC it's just a five nanometer long so quite small organisms but it's very

important for human because we are getting many many product from surco my sister Missy and this organism is also

quite good system for their to understand their our cell so called you character cell so whole German sequence

and we have many so huge database on this maker organism when I come back from their United States from

Rockefeller University my boss said you can work on East any subject so I wondered what is the quite interesting

thing for me and I'm not a competitive person so I don't want I do not want work work in the familiar popular field

so I interested in the gesture this quite simple organelle bacio when I at that time I was in a biker botany Cal

Department and so I interested in their back yo because at that time many people so just Barker is just a garbage trash

in the cell but why plant cell has sold ROG back you all of you probably know from their elementary school or middle

school or high school year on prank cell has a large back you but you probably not so many people

think about why Prancer has so much so big vacuum so I won't say it is a week difference you know and to understand

it's a very big difference and if we have time I won't discuss about this later so I think there are many many

things to be answered in nature so I about 10 years I worked on their back your membrane and I found Baku is not

inert Organa it it takes lots of amino acid or a calcium or many many kind of blur as my tears are story I mean this

organelle and the driving force is a gradient important proton gliding gradient is made by new type of protein

atps brought so called blue type a dps then in 1988 i got a small ab just i started by myself at the tokyo

university and at that time i want to change my subject to another new subject and i want to understand the Riddick

function of East Parker because as I said Barker is a ascetic compartment but be a we type a TPS it was known Baku

contains the rarest kind of hydrolytic enzymes protonation protein degradation nuclear and nucleolus tidus man osiris

phosphatase so that must be the ridic compartment the validation compartment so I thought that might be a homologous

by a racism so-called lysosome in mammals I don't need so as you heard from their first talk today all protein

are the made by beer the genetic code in DNA to RNA to protein and these two decays we learned all proteins are made

in the site present and they are transported a proper compartment or many kind of their agha narrower so we learn

how protein Roka is appropriate this just figured we don't know about their fate of the each protein each

protein has a quite distinct lifetime from fuse a few minutes to three months or more so

before I go to there my work our work I want to say several general scheme of the protein degradation we did our cell

body and I when I was a I had a biology class at Tokyo University always I started my talk my lecture from this

question how many blood cells are made per hour only one second in our body you can easily calculate within few minutes

the answer is that Brazil is made three million cells per second and how about hemoglobin hemoglobin is er it was in

let Brad sell it bound to oxygen and this kind of molecule one times 10 to the 15th molecule per second we are

making that me exactly means this number of cells and proteins are degraded and okay construction

it seems more positive and the disruption the disruption is a negative way but in our ours for example this is

a Tokyo University campus in the spring you can see leaves develop in summer you can see lots of green leaves and

actively photosynthesis and in autumn you can enjoy these yellow color ribs you know these kind of

every year and but here why these leaves turn yellow that means older claw pressed the green pigment straw

photosynthetic machinery is degraded and all the amino acid transported to the trunk and another example is this is a

rice field in the summer you can see green leaves by sunshine these reefs actively Fateh Singh said starch and in

autumn you can see yellow leaves again exactly same thing happened here their reason leaves turn yellow that means all

the protein on the Andreas are degraded completely and all the amino acid go to the this grain of the

rice so for rice any degradation is very important to make next generation these grains so actually degradation is not

negative way it's very important and another example we are making every day about two to three hundred grams of

protein but we are taking 70 to 80 grams of protein as a food and if can we make whole these amino acid protein turn to

the amine acid it's simply 70 grams is not sufficient for make this kind of protein so just to

this simple number suggest over protein in our body degrade and most of them in acid come from there our

protein our own protein so we our system is adjust equilibrium state of their protein synthesis and degradation

so actually life is a equal state between synthesis and degradation protein so to my supplies almost every

three months almost almost all protein in our bodies is replaced so recycling is a really essential for life there

first like through in intracellular protein devaluation was made by Christian de

Duve version so what is he found her small membrane organelles called lysosome that contains a little enzymes

within and then look for a group including Christian deduced group analyser how protein get into the

lysosome and they found they named their own sites Erick protein transfer to the vacuum to the lysosome that means a self

eating process Otto Otto that this is a he coined the name of the process as Otto AG since then they're another

very important protein degradation system was found that these are victim protostomes system that is very

important to degrade a specific protein just tagging their a small protein evicting

and I'm degrade by huge protests on protests home and this is very important for various kind of biological system

regulation but lysosome or a buck your system almost nothing was known about me molecular mechanism over genes involved

in that process as I said I was working on long time under vacu vacuum and I want to know is it's

very there Hritik compartment within self because this is a cell a compartment and contains very notably

degrading enzyme so the questions I had is when and what and how cytosolic protein come across this back your

membrane and degraded first I thought what is the best condition to work with is so if you have a sufficient nutrient

he so can grow just this kind of a budding but if nitrogen source I mean acid the source of them in acid

nitrogen starvation induce this kind would quite dramatic change of the cell may OSIS occurs and these diploid cell

forms for spores that this process is very important for genetic analysis of the

cell so this quite big change within cell should be done without any supply of the nutrient outside so this process

should be required the roots of own protein degradation so I sought nitrogen starvation must be might be very good

condition to study the protein degradation and I thought if I see there re step of their expiration under

microscope I might see some something under microscope but not unfortunately I couldn't see anything under light

microscope one day I thought if I use the protein protein is the efficient mutant cell that was already constructed

by Elizabeth Jones in the United States I got these kind of their triple pack your protein is the efficient mutant

cell and if I use this cell and the starvation condition if Blackie the structure over something delivered to

the vacuole to be degraded might be seeing on the right microscope okay this is a phenomena found and the growing

cell or just the zero time used has a vacuole but nothing inside in the back you but after three hours

and the starvation condition you can see this kind of swagger structures moving around in the side soul and to my

supplies almost a whole selves has this kind of structure so this might be quite interesting phenomena and so this was a

starting point of the whole work still in my lab many people just observing this phenomena so this is a starting

point and always we are asking what what why this can happen under starvation conditions this is a time course of

their phenomena I showed after 30 minutes we can see this kind of small structure appeared in the vacuum and

gradually increase means after three hours almost all vacuole occupied this kind of structure so we started the

electron microscopic analysis of this phenomena mr. Bober my good collaborator worked on this subject and this is a

three hours starvation stabbed cell you can see within vacu this kind of structure in

this small pocket contains and this higher high magnification image you can see the structure is a single membrane

bound you can see these black dots are ribosome and you can easily think about the inside of there

this structure is a really a portion beside the polar right front here so from these

picture we sold nitrogen starvation in Eustis kind of blood a structure for delivering sight browser component

London Rea non selectively to the vacuum you can see various kind of cytosolic structure everything de structure we

named this as an old Hajek body data here you can see a la fere here and many electron microscopic study we found

this is a barrio next to the back you you can see this kind of cup shape membrane sack appear just end up being a

portion of site soul and this is a already enclosed double membrane structure autofocus ohm in east and

those these double membrane structure outer membrane of the government structure is continuous to them about

your membrane and the inner membrane is just showing here and these are the inner membrane structure released in the

back us up we so called automatic abilities so as you see here here's a just form a home forming a double

government membrane structure here you can see these structure contains ribosome ribosome is already quite there

super quite big structure but you can see mitochondria here and here and here

that means the autophagy is quite unique system degrade the whole organelle by end up being a portion was height so

so it is quite different strategy if we compare their evicting protozoan system that strictly recognized one

protein that should be degraded by sophisticated enzyme system but autophagy is quite bulk is good for bulk

degradation of own such a component in the lytic compartment so almost thirty years ago we draw this kind of picture

other automatic processing in yeast when his cell faced various kind of their nutrient starvation suddenly this kind

of small membrane sac appeared in elongate and former double memory structure of the ribosome and odd

phagosome target to the barrio and fusion event occurred and in the membrane structure is our latest in the

back yo sup but these structure is so quickly decedent disintegrated in wild-type cell within half minutes or so

so nobody found this kind of structure I used the backer protein is deficient the mutant mutant I could follow the

automatic process as accumulation of all tragic abilities within barrio so next we tried of obviously we try to

analyze their machine mechanism of load of in East to by genetic approach to make by isolating the auto fuzzy

defective mutant if we could get their hot fuzzy defective mutant we can easily get the genes required for sir

Auto fudgy in East but we didn't know anything about what is the phenotype of their auto fudgy defective mutant in

yeast so we started the microscopic screening no Auto fasiq bodies accumulation within vacuum just under

microscope one by one and when my graduates the first graduate student succeeded isolate only one auto fuzzy

defective Milton at that time we named we called a PG 1 now we named a TG 1 Newton but buckaroo is a quite complex

membrane phenomena so we should have more ATG genes but a TG 1 mutant is quite normal

under nutrient rich condition standard but east medium but we found a TG 1 mutant can not survive for a long time

nitrogen starvation yes after 2 days ATG mutant start to die so I assume this loss of viability phenotype is called

visor or defect in Auto foggy so we tried loss of ability same time as the first

screening and we then we again use Michael's right microscopic screening the mutant like the automatic body using

vacuum this way we could easily get there about hundred or fuzzy defective mutant gesture genetic analysis we could

get fourteen eighty genes as a first screening then now we call and we say eighteen eighty genes I essential for

there out for confirmation so the first screening was could be quite the efficient that helped a lot

for as other analysis and next question what is a disease but kind of protein is coded by eighty genes we started the

cloning or a disease sequencing of a disease and identification of eighty g proteins but first three or five eighty

genes or novel gene i we couldn't say anything about the function of 80 genes from that kind of analysis so it was

really hard time for us but I have no time people called in detail but first we found quite interesting me

Ubik ting autophagy equals two kinds of you became like conjugation system ATG twelve is much bigger than the built-in

but it's a activated by an enzyme to transfer to another protein by this kindness so it's a intermediate and

finally former you became like I so peptide bond formation with 85 and interesting more interesting Lear we

have warmed another direction like floating within our genes 88 is the c-terminal arginine is removed by this

protein is and grazing sitter mobilizing is activated again the same even enzyme transferred to 83 and to our supplies

ATG 8 is not conjugated with the protein but it's a membrane phospholipid for specially terminal I mean was a target

of 80 g and within several years in their National Institute basic biology I moved

we could found many many new finding about ATG genes initiation is a tall kinase is a very important upstream the

greater of their prophecy because if other not my sins I heaved out talkin even in quite nutrient which condition

Auto Hodges studied and I will show you a little bit about this initial step though although also house 'information

data within several years eighteen eighty g genes were classified into this kind of reaction subgroups ATG one is a

protein kinase a DG certain is a important regulator of the initial step of the whole ecology and we found

pi3 kinase it's a membrane phospholipid you know you know you know stole phosphate is phosphorylated by this

kinase pH D P is important generation is very important for the membrane dynamics and as I said these

two convicting like conjugation is required for the auto fudgy and a tg9 is only membrane brought multimeric

membrane spanning floating among adhesions and yet we cannot say exactly the function of this complex 82 and 83

18 complex so 80 G's just are required to further Auto confirmation we found and when I

was lucky in an IV B because tumultuous amore and noble mizushima they joined our lab and they started there it is

genes in mammals and to post postdoc and graduates didn't start work on plant ATG gene and we found ATG genes are well

conserved from yeast to human and clung so I think identification of 80 genes but completely change the landscape of

Auto fudgie research the reason is ok before as I said moto is only detectable by under by using electron

microscopy but now mobile mizushima Mader GFP lc3 it's a horrible way to g8 in yeast and tag GFP and expressed whole

body translating car mouse if we analyze this mouse we can say where and how much Auto Howsam is formed just underwrite a

fluorescence microscope this is a muscle and this is a eber cell and this is the fertilized egg so we don't need the

electron microscopy anymore and as I see you see this is a fibroblast and as night starvation you can see lots of old

photos of mrs. form so this is a actually degradation Bible prophecy so now we know Oh - Fozzie has a major to

lows one is oh of course nutrient recycling that's very important for survival mechanism and another important

aspect of the toughest elimination of excessive or harmful materials from cytoplasm so these are quite many people

interested me that this selective degradation of harm mitochondria lysosome sometimes a year

and a portion of my nucleus and also in basic bacteria or virus particle B or tofurkey so but we our group interest in

the how ATG gene function photo confirmation and we found this is a now we fluorescence microscope technique was

very developed so we can see eighteen seventeen how that behavior in the cytosol within sites all eighteen

seventeen it's a actually a dimer of ATG 17:30 a 29:31 just moving around in the side so and under starvation condition

portion of EDS having King stick to the discernible dot structure we named prey auto focus

on structure this is the site of the odd formation so we think under for example nitrogen starvation ATG 180 13 29 31 29

31 complex is formed next to the barrio that we call past and then 80 G's nine bicycle come and a PA pi3 kinase joined

to the past and ATT to 18 complex is joining and finally to conjugation system is required for the irrigation

step of the isolation membrane and but just I we we showed this every protein is adjustor Socko but now we by

collaborating with ER dr. flaw in Avakian Oda's group we cannot determine the three-dimensional structure most ATG

protein that helped a lot to understand the molecular detail of the autosum formation step just I won't show you the

step how early step is okay with himself Oh so we

what okay now okay I just after this talk I will show his happy movie okay now we're

working on just a fundamental question I asked first what these are what is when and how cytosolic materials

degraded by auto fudgie we need lots of works what is our induction condition and what these are signal transduction

for induction and we want to know about their really target of their auto fancy for example what kind of protein

decorated to the baculum autophagy Ottawa so it's not we I believe it's not simply bulk non-selective degradation

some protein are preferentially degraded by Auto physique and I think there are many many moles of water veggie still we

should work disc and question also not so many people interested in degradation process and degradation product by Auto

Fuji so we want to know about this process and so without this kind of data we cannot say really the physiological

meaning of Auto fashi one example we issue I was shown the Auto fuzzy was solved the protein degradation system

but recently we found lots of RNA you see the lots of our live assortment is taken up into the barbecue beautifu G

so this RNA should be degraded and we found the enzymatic process of RNA degradation with about you

t2 type nuclear nucleus is important and three prime nucleotide type is formed and nucleotide come out from these

barrio and nucleoside immediately degraded to the base under rising like libels and this base are almost

completely come out from the cell so this is not just a simply recycling but nucleotide metabolism is very change

during the this kind of acceleration so this is a paper of the facilities research I started out the fuzzy work

here 1988 at that time only 20 or 10 papers per year and not so many people interested in the protein degradation

and Otto Hoshi was not popular in even cell biologists and we found the Otto fudgy in his first paper appeared here

and we found several interesting gene function of genes but it as you see here now

Otto field is really become quite popular field in cell biology shown here but I want to say it takes

10 or 20 years to become this kind of hot field in biology so I won't say there are many people young people I

want them you you can work with this kind of really popular feel if you can really get you feel you you will have a

good result but these these are not a competitive field but I think some challenging person start something else

somewhere here and we make her discover new field so just 27 years about say 29 or 30 years of my autophagy research was

really not so flat but many was helped by many accident and wonderful occasion I really sang closer good collaborator

and friends and my I want to emphasize my whole work was just a cure curiously driven research I never thought the

autophagy filled with be related to the cancer or many many kind diseases there that quite now popular film in auto

fudgie research but I was so I want to emphasize the curiosity driven research is very important to that in the end

this is a message for young generation from me okay that's all

[Applause]