レポート課題 6 ～ 7. （最終締め切り： 1 月 29 日）以下について、講義を通じて理解したこと（あるいは調べたこと）をまとめよ。

6. 細胞内のタンパク質ターゲティングの仕組み：真核細胞には沢山のコンパートメントがあるが、それぞれに送られるべきタンパク質が選別され送られる仕組みについて（特に ER translocon の場合、または、それ以外のオルガネラについて ) 。

7. 膜小胞形成および標的膜との融合の仕組みについて。G-proteins, Coat, SNARE などの役割を理解する。

ER 膜についている Polysome

１／３のタンパク質はこの経路で！

Vesicle budding-fusion

Vesicle flowor

Membrane flow（小胞輸送＝膜動輸

送）Budding→FusionIn vitro 再構成系

でも再現！！

Many membrane shaping proteins!

末端の 3Glc はグルコシダーゼ I and II (GI and GII) によって切り取られる。 GII と UDP-Glc glucosyltransferase (UGGT) の反応サイクル(chaperone との結合・解離を伴う ) の間に folding が進行する。やがて、タイマーとして働く -1,2-mannnosidase によって (Man)8(GlcNAc)2 となったもののうち、 UGGT に認識されない正しい conformation に到達したものだけが COP II 小胞へと選抜され Golgi へ。どうしても目標に到達できないものは最終的に Glc(Man)8(GlcNAc)2 となって ( 赤矢印 ) 下図分解系へ。

正しい形でないものを見わけて回収

Among a large amount of newly synthesized proteins in the ER, 30% of them do not reach a correctly folded conformation.

熱に不安定な蛍光タンパク質を発現39.5℃ では ER にとどまる (Quality Control!)10℃ にすると、 folding して Exit site に集まる（温度が低いので Golgi へは移行しにくい）20℃ では Golgi へ

酵母のミュータントで詳細が解明される

sec mutants of yeast (and E. coli)

Movies: Sec_Vesicle.mov

Sec_tubule_MT.mov

ER to Golgi transport

tER=ERES/ERGICを経由して？

transitional ER ＝ ER exit site/ER-Golgi intermediate

compartment

22: 471–478, 2010

Exit sites

ERGIC

Clathrin-Coated Vesicle

Trans Golgi Network

Golgi Cisternae

Trans

Medial

CisER-Golgi Intermediate Compartment≒Vesicular tubular clusters (VTC)

COPII Vesicle

Exit site of ER (tER)

Vesicular tubular clusters

動物は小胞を運ぶ、植物はGolgiを運ぶ？

COPII-mediated traffic in plants

Trends in Plant Science 15: 522–528, 2010

ER Exit Site とGolgi の距離が近い＝ ERGIC がない？

Exit site で何が起こっているか蛋白質核酸酵素　 Vol. 49 No. 7 (2004) 910

SNARE, Rab などの specificity factor もリクルートされる

70 ～ 90 nm vesicle

Sar1 GTPase (secretion-associated and Ras-

related protein)

Sar1-GTP→GDP

1. Coat

• Activation of the small GTPase Sar1 through its guanine exchange factor (GEF ) Sec12 (=scaffold).

• GDP/GTP exchange leads to the exposure of an N-terminal amphipathic helix of Sar1, with which it can insert into the ER membrane.

• This insertion causes membrane deformation and is ultimately required for membrane fission.

• Directly interacting with Sec23, Sar1 recruits the heterodimer Sec23–Sec24. (The majority of cargo is captured through interaction with Sec24, which exhibits multiple independent cargo binding sites. Sec23 is a GAP for Sar1! )

• After the formation of pre-budding complex (with cargos incorporated and membrane deformed [curvature!]), the outer layer of the coat (heterotetramer Sec13–Sec31) is recruited to the ER membrane.

• Sec13–Sec31 can self-assemble into cage like structures with a cuboctahedral geometry.

• The crystal structure reveals relatively weak inter-subunit interfaces and a geometry that could allow greater flexibility of the COPII coat, compared to other coats such as clathrin, in order to accommodate cargo of different shapes.

• Soon after budding, COPII vesicles uncoat due to the GTP hydrolysis by Sar1. The very slow intrinsic GTP hydrolysis rate of Sar1 is accelerated in two steps: first through the interaction with Sec23 acting as GTPase-activating protein for Sar1, and second through the binding of Sec13–Sec31 to the pre-budding complex, which increases Sec23-mediated GAP activity by an order of magnitude, presumably by optimising the interaction of Sec23 with Sar1.

• While inherent instability could present a problem with regard to stabilization of the COPII coat, the ongoing presence of Sec12 will provide a continuing supply of Sar1–GTP and cargo–coat interactions are known to stabilize the pre-budding complex even in the presence of ongoing GTP hydrolysis by Sar1.

• This dynamic interplay between coat assembly and disassembly clearly has important implications for budding.

• Sar1, Sec23–Sec24, and Sec13–Sec31 are the minimal machinery required to reconstitute COPII-dependent budding in vitro.

• GTP-dependent budding requires Sec12 in addition. In vivo, multiple other factors are likely to play key roles. Notably, COPII budding in mammalian cells is ATP-dependent and sensitive to protein kinase inhibitors.

Exit Site Membrane ( くぼみ !)|

Coat|

ERGIC Compartment

FEBS Letters 583: 3796-3803, 2009

Sec16 はSec12 と共局在する足場Scaffold

ER-Exit Site(ERES or tER)

ribosome-free ER subdomains

approximately 0.5 m in diameter

ERGIC (ER-Golgi Intermediate

compartment)or

VTC (vesicular tubular cluster)

Cis-Golgi

Journal of Cell Science 119, 2173-2183 (2006)

ERGIC marker ERGIC-53 immunodetected. 糖鎖を認識して、可溶性のカーゴタンパクを集める！

Exit site と ERGIC は完全には重ならないが近接する

FIGURE 3 | The ER–Golgi interface and ERES have a distinct organization in mammals and plants.

Nature Reviews Molecular Cell Biology 14, 382-392 (June 2013)

FIGURE 4 | Cellular architecture contributes to the ER–Golgi organization and positioning of ERES in mammalian and plant cells.

Golgi から先はかなり複雑

小胞 Vesicle は主に出芽の際のコートの種類で

分類される曲面を作る役割１

細胞工学 Vol. 25 No. 11 (2006) ARF によるメンブレントラフィックの調節

Anterograde & retrograde flows

3 種の小胞とそれを形成するコート　（ Vesicles: タンパク質と脂質両方を運

ぶ）

Secretory pathway － vesicle traffic

RetrogradeCOPI vesicles

COPII vesiclesAnterograde

「細胞の分子生物学」ニュートンプレス

Endosome

Clathrin coatほかの二つは寿命が短かく、特徴的な構造がないので、最近になって発見された

主に細胞膜Cargo 膜タンパクを認識・集積して

形成比較的すぐに解離

MBC Figure 13-8.

主な Coat は３種類これでは足らない！

何が特異性を決めているか？？Cargo に対してはGTPase + Adapter膜 ( 目的地 ) 特異性では付加される SNARE, Tether, Rab( これもGTPase) and its effectors（ >60 Rabs and >30 SNAREs)

細胞内輸送がわかる　「小胞体 - ゴルジ体間輸送」

1. Budding, 2. uncoating, targeting － 3. tethering － docking, priming & 4. fusion

Cargo の選択があるかどうかが大問題

Coat の役割２Cargo recruitment/selection

可溶性タンパク：膜タンパクリセプタを介して膜タンパク：直接認識

Exit signal

Trends in Plant Science 15: 522–528, 2010

Protein Sorting Receptors in the Early Secretory Pathway

Annual Review of Biochemistry 79: 777-802 (2010)

Figure 2  Model for coat protein complex II (COPII)-dependent cargo selection and vesicle formation.

代表的な Cargo receptorERGIC-53: mannose-binding lectin, required for the export of several cargos from the ER as well as (along with other cargo receptors) for maintaining the structural integrity of the intermediate compartment.

Cargo receptors, an example

ERGIC-53: mannose-binding lectin, required for the export of several cargoes from the ER as well as (along with other cargo receptors) for maintaining the structural integrity of the intermediate compartment. Mutations in the ERGIC-53 gene cause autosomal recessive bleeding disorders resulting from the inability to package the blood clotting factors V and VIII.ERGIC-53 interacts directly with Sec23 leading to the recruitment of ERGIC-53/cargo complexes into COPII vesicles. After reaching the ERGIC, the cargo dissociates from the complex and ERGIC-53 is recycled back to the ER.

Figure 4  Model showing structures and membrane topologies of sorting receptors in the early secretory pathway.

COPI vesicleで戻すreceptor

Sorting receptors

aAbbreviations: HDEL, a C-terminal amino acid sequence; dm, Drosophila melanogaster; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; GPI, glycophosphatidylinositol; I-L-V, an amino acid sequence motif; hs, Homo sapiens; KDEL, an amino acid sequence; MCFD2, multiple coagulation factor deficiency 2; sc, Saccharomyces cerevisiae.

Retention of incompletely folded

proteinsシャペロンが結合しているので、receptor に認識されない！

Sar1=G-protein を足場に

Cargo と Coat が集合する それ以外に、後の小胞輸送・融合に関わる多数の因子も同時に、

あるいは直後に

Cargo の選別小胞の出芽

Coat が完成（して出芽が成功）すれば、すぐに Sar1 は抜け落ちる？ Clathrin coat では、 Coat も比較的すぐに外れる

COPII coat in ER-Golgi transportSar1 (G-protein) and its GEF Sec12

Coat 成分に GAP

ER exit sites – Localization and control of COPII vesicle formation

FEBS Letters 583: 3796-3803, 2009

足場 1/Sar1 GEF

足場 2

AdaptorOuter coat

Sec12 と Sec16 が足場

Volume 14, April 2004, Pages 147-153

GTP 結合型になると膜アンカー（赤）が飛び出す！

Unhydrolyzable analogue of GTP

Sar1 Activated by Sec12 (its GEF) and anchored to membrane by its own N-terminal (above)

Trends in Cell Biology 13: 59-60, 2003

親水性面

Activation of Sar1 by its GEF Sec12

Nature Rev Mol Cell Biol 7: 727, 2006

Prebudding complex:

Sar1+Sec23/24（アダプター）

膜にアンカーしたSar1 を起点に曲面を作る！Trends in Cell Biology 13: 59-60, 2003

A & B Cargo-binding sitesVolume 14, Issue 2 , April 2004, Pages 147-153

Adaptor 別の役割Sec23: Sar1 GAP

Sec24: Cargo/Receptor の認識

A cargo, transport protein (red: CFTR) with diacidic motif (DXE/DXD, green)

Cargo も必要！！

さらにその上にCoat の主要部Sec13/31 がかぶさる！

cuboctahedral cage 　 14 面体（６＋８）立方体の頂点を落とした形　

igure 2 | The architecture of the COPII cage facilitates transport of diverse cargo.

Nature Reviews Molecular Cell Biology 14, 382-392 (June 2013)

Outer Coat – Sec13/Sec31

7: 727-738, 2006Zig-zag -helices

- プロペラ と -ソレノイド

β -propeller: β シートでできた羽根 4-8枚が中央の軸の周りを円錐状に取り囲んだ形をしている。それぞれの羽根は4 つほどの逆平行 β シートからできていて、 1 つ目と 4 つ目の β シートはほぼ垂直になるくらいにねじれている。

インフルエンザウイルスの持つ酵素であるウイルス・ノイラミニダーゼは 6枚羽根の βプロペラ

http://www.calvin.edu/academic/chemistry/faculty/arnoys/arnoys-chem324-beta%20propeller.html

構造がうまく組み上がると、最後には， GAP (Sec23) が働き脱コー （ﾄ time

switch ！）

Volume 14, Issue 2 , April 2004, Pages 147-153

水

GTP analogueSec23 の R側鎖が挿入される

Mg2+

脱コートする前に、 Sar1 は(Sec23/24, Sec13/31 よりも )早く　 turnover している！

遺伝病 cranio-lenticulo-sutural dysplasia (CLSD)頭蓋・水晶体・縫合異形成症 http://www.rightdiagnosis.com/c/craniolenticulosutural_dysplasia/intro.htm

主にコラーゲンの分泌異常による

Sec23A遺伝子変異によりCOPII 小胞がうまくできず、 ER から突出する構造のみ　B-D: heterozygous

(unaffected) cellsE-G: homozygous mutant cell (一部Coat も見える：F,G)

小胞体ストレス応答を介した骨軟骨形成制御生化学 84: 18-29, 2012参照！

Clathrin（アームの部分は短いゆがんだ αヘリックス

が作るチューブ構

造）と似てい

るCoat の外層は共通の構造で、 shell殻構造を作る

Clathrin Coat （ Cage 籠）

COPII and Clathrin cages

COPII and Clathrin

nature04339-s5.mov

36 Triskelionの構造

曲率の異なる構造を作りう

る

外殻

内層＝アダプター G-protein, Cargo, 外殻との界面

Arf なし

内層＝アダプター G-protein, Cargo, Coat shell との界面

Structural & Evolutionary aspects

NCBI Books: Development

Coat GTPases: Sar1 and Arfs(ADP ribosylation factor)

外殻の構造は少しずつ異なるが基本のモジュール構造は同じ

COPII に戻って：ほぼ一定のサイズの小胞ができあがる

　 70 ～ 90 nm

Volume 14, Issue 2 , April 2004, Pages 147-153

Main coat=Sec13/31 complex

Prebudding complex on a 60 nm sphere

In vitro reconstituted buds

Sec23/Sec24 complex=adaptor

Sar1/Adaptor (Sec23/24) unit の数辺の数の二倍

• 60 nm 径（右図）で24辺、 48個

• 90 nm となると、下図右端で～ 200個

うまく数えられません！

nature04339-s5.mov

64 100

いくつかの問題

1.ｔ ER (ERES) はどのようにできるか2. Cargo selection があるか（多分ある．でなければ， Bulk flow で間違って送られたものを COP I 小胞で戻すのみ．）

But how and to what extent??

3. Sar1 GTP 加水分解の役割（ fission自体にも関与？）

1. Sec16 と Cargo が Exit site を作る？

Sec23-YFP で可視化

De novo formation, division & fusion

EMBO reports 4: 210-217, 2003移動する　増える

1 分間隔で観察

Sec12 は mobile!• Soderholm1.mov Pichia pastoris

Fluorescent recovery after photobleaching (FRAP)

蛍光は 35% に Dev. Cell 6: 649-659, 2004

鏃部分を消光、蛍光が戻っている！

Sec12 を過剰に発現させると，細胞質に（ 2 ・ 3段目）(saturation)

他と相互作用してExit site に集まる

発現停止後の時間

過剰

過剰

S. cerevisiae ではSec12 は ER 全体に広がっている。 Pichia の Sec12を発現させても同様の挙動。（ Pichia の細胞では，それ自身でなく，他と相互作用して集まる。）逆に， S. cerevisiaeの Sec12 を Pichia に入れても集まらないので，構造特異性・種特異性は高い。

3. Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission

JCB 171: 919-924

N- 末端の挿入自体がリポソームに影響人工膜Liposome+Sar1 N-term

(C) Sar1-GTP is capable of constricting liposome membranes. Liposomes (80–100 nm, DOPC and DLPA, 80/20 mol percent) were incubated in buffer (control; a), 10 µM Sar1-GDP (Sar1-T39N, b), 15 µM Sar1-GTP (Sar1-H79G, c), or 10 µM 9-Sar1 (d) mutants with GTP or GDP for 1 h at 37°C. (D) A gallery of Sar1-GTP–induced tubulating (a–d) and fused (e and f) DOPC/DLPA liposomes. E) Cholesterol/DOPC/DLPA (20/75/5 mol percent) liposomes (100–120 nm) were incubated as described above in the absence or presence of Sar1-GTP as indicated. (F) A gallery of tubulating cholesterol/DOPC/DLPA liposomes deformed during incubations with Sar1-GTP. Bars, 100 nm.

チューブ構造が作られる！

GTP-S (unhydrolyzable)

Sar1 Sec23

Sec13 Sec31

Permeabi-lized cells

GTP 加水分解を阻害すると、小胞まで進まない

GTPase活性は膜の曲率が高くなると活性化される（別の Coat GTPase Arf1 ）

Nature 426: 507, 2003

Trp残基の蛍光：疎水環境で強い

フィルター孔を通すことで、サイズの異なる Liposome をつくる

サイズが小さい方が GTP 加水分解が早い

光散乱でコート形成解離を見る

小さいリポソームほど早く解離する。と言うより turnover! している

ダイナミックなコート　トレッドミリング

COPI-Arf のみの状況かもしれないが

Treadmilling

COPII function

(i) Exit site で Cargo を集める(ii) 出芽後に必要な成分 SNAREs, Rabs,

tethers, motor protein などを複合体としてリクルートする

(iii) 膜の出芽・小胞化

Sar1/Sec23/Sec24 のターンオーバー（入れ替わり）＝ 1.1/3.7/3.9 s (Curr Biol 16: 173, 2008)

Cold Spring Harb Perspect Biol February 2013;5:a013367The highly conserved

COPII coat complex sortscargo from the endoplasmicreticulum and targets it to the Golgi

2. RabGTPase

Vesicle flowの総元締め

Huge number of effectors!

The directionality and fidelity of coat protein complex II (COPII) vesicle fusion is mediated by concerted action of RAB GTPases, tethering factors and integral membrane SNARE proteins. In mammalian cells, the vesicle targeting stage depends on RAB1 (Ref. 30) and the extended coiled-coil domain tethering factors p115, GM130 (cis-Golgi matrix of 130 kDa) and GRASP65 (Golgi reassembly-stacking protein of 65 kDa)137, 138 as well as the 170 kDa multisubunit TRAPPI (transport protein particle I) tethering complex (comprising BET3 (blocked early in transport 3), BET5, TRS20 (TRAPP subunit 20 (also known as Sedlin), TRS23, TRS31 and TRS33) that exerts guanine nucleotide exchange factor (GEF) activity towards RAB1 (Refs 139,140). RAB1·GTP recruits p115 and tethers vesicles to acceptor membranes. Therefore, the activation of RAB1 on acceptor membranes by TRAPPI may generate a localized signal to tether COPII vesicles.

Nature Reviews Molecular Cell Biology 14, 382-392 (June 2013)

Golgi体が形を保っている 1 つの原因

Golgins – Tethering proteins 　（５）

BBA 1744: 383, 2005

GRASP protein with an N-terminal myristoyl group

The BET3 subunit of TRAPPI also binds directly to the SEC23 subunit of COPII and can tether COPII vesicles at a close distance31. Fusion of COPII-tethered vesicles depends on a set of four tail-anchored integral membrane SNARE proteins named syntaxin 5, membrin (also known as GOSR2), BET1 and SEC22B141, 142, 143. SNARE proteins contain a conserved membrane-proximal heptad repeat sequence known as the SNARE motif, and trans-assembly of cognate sets of SNARE proteins from donor and acceptor membranes into four-helix bundles drives bilayer fusion144, 145. Therefore, trans-assembly of syntaxin 5, membrin, BET1 and SEC22B between tethered COPII vesicles and Golgi acceptor membranes catalyses fusion. The conserved syntaxin 5-binding protein SLY1 is also required for this vesicle fusion step146 and may serve to coordinate the vesicle tethering and fusion stages. Regulation at these multiple stages in COPII vesicle tethering and fusion may have crucial roles in determining ER–Golgi morphology and levels of coated transport intermediates, but is relatively unexplored.

Coiled-coil が膜を接着させ、融合させる

水を排除

Molecular Biology of the Cell. 4th edition.

The Highly Conserved COPII Coat Complex Sorts Cargo from the Endoplasmic Reticulum and Targets It to the Golgi Christopher Lord, Susan Ferro-Novick, and Elizabeth A. MillerCold Spring Harb Perspect Biol February 2013;5:a013367

Tether: TRAPPIYpt1 (Rab) activatedUso1 (another tether) recruited

Then SNAREs come!

S/T kinasephosphorylates Sec23/Sec24

ER export signal

• C-terminal FF, di-acidic and di-hydrophobic motifs

選別に関わるのは，膜貫通型の積み荷受容体 (cargo receptor) ERGIC-53や p24

例外は多数

(GPI-anchored proteins--Caveolae)

少なくとも大きすぎで COP II vesicle に入りきらないものもある

• Procollagen• Chylomicron (270 nm!) Sec23 の代わりに Lst1/Iss1??

COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicleJCS 2003 116: 415-427. in vitro 系

Current Opinion in Cell Biology 17: 345-352, 2005

Procollagen fiber に結合する TANGO1 の助けで大きな小胞が作られるSec23/24 に結合することで Sec31/13 の結合を遅らせ、 bud のサイズを大きくする

EMBO J. 2011 August 31; 30(17): 3475–3480

http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3181478_emboj2011255f3.jpg

N- 末端がミリストイル化

Retrieval of ER resident proteinswith KDEL signal

V-ATPase making low lumen pHKDEL receptor がこの環境で相手を認識・結合 ER へ戻る　

　　　　　　　　　

別の図　 MCB

Retrograde transport via COPI vesicle

• 選別シグナル : ジリジン（ --ＫＫＸＸ）モチーフ

KDEL レセプター , p24 ファミリー , ERGIC-53 (ER export signal をも持つ ) などが持っているジスルフィドイソメラーゼ (Protein disulfide

isomerase ： PDI ） や分子シャペロン BiP は小胞体内腔で機能するタンパク質であり，いずれもＣ末端に KDEL （ Lys-Asp-Glu-Leu ）モチーフをもつ．これらの可溶性タンパク質は KDELレセプターによって，小胞体へ回収される．

Box 2 | The COPI coat complex and retrograde transport

14, 382-392 (June 2013)

http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=2228377_418_2007_363_Fig2_HTML.jpg

Golgin (p115), GM130, Giantin,

COPII 小胞が融合してVTC

VTC とcGolgi の繋留

COPI 小胞と cGolgi

3. SNARE

Molecular Biology of the Cell. 4th edition.

Golgi!

Golgi tomography

• BBA 2005 Volume 1744, Issue 3 p. 273 by Marsh BJ

Fig. 5c!!

http://www.sciencedirect.com/science/article/pii/S0167488905000601#MMCvFirst

Golgi での糖鎖修飾

=TGN

Golgi

糖鎖修飾と積荷の選別ステーション

ゴルジのいろいろ

A goblet cell of the small intestine

A cultured fibroblast and a plant cell

Saccharomyces では Golgi各槽板がばらばら！

Golgi体が形を保っている 1 つの原因

Golgins – Tethering proteins 　（５）

BBA 1744: 383, 2005

GRASP protein with an N-terminal myristoyl group

GRASP: Golgi reassembly stacking proteinGRIP (golgin-97, RanBP2alpha, Imh1p and p230/golgin-245) domainGRAB (GRIP-related Arf-binding)

Vesicular transport model and

Cisternal maturation model

積み荷を含んだ小胞が移動するか，膜が移動し，固有成分は逆方向に移動する？

Golgi槽板の成熟

Nature 441, 1002-1006 (22 June 2006)Movie 1a:  Sec7p-GFP labeling.  Shown are projected images of Golgi cisternae labeled with Sec7p‑GFP.  The z‑stacks were taken at 1.5 s intervals.  The arrows and arrowheads mark four Golgi cisternae that acquired and then lost fluorescence.  This movie is labeled “D” in Table S1. Movie 1b:  Sec7p-GFP labeling, edited.  The 4D dataset for Movie 1a was edited to show only the cisternae marked by the arrows and arrowheads.  Movie 2a:  Sys1p-GFP and Sec7p-DsRed dual labeling.  Shown are projected images of Golgi cisternae labeled with Sys1p‑GFP and Sec7p‑DsRed.  The z‑stacks were taken at 3 s intervals.  The arrow and arrowhead mark two Golgi cisternae that acquired green and red fluorescence almost simultaneously, then transiently contained both marker proteins, and then lost green and red fluorescence almost simultaneously. Movie 2b:  Sys1p-GFP and Sec7p-DsRed dual labeling, edited.  The 4D dataset for Movie 2a was edited to show only the cisternae marked by the arrow and arrowhead.

Movie 3a:  GFP-Vrg4p and Sec7p-DsRed dual labeling.  Shown are projected images of Golgi cisternae labeled with GFP‑Vrg4p and Sec7p‑DsRed.  The z‑stacks were taken at 3 s intervals.  The arrow and arrowhead mark two Golgi cisternae that acquired green fluorescence, then lost green fluorescence while acquiring red fluorescence, and then lost red fluorescence. Movie 3b:  GFP-Vrg4p and Sec7p-DsRed dual labeling, edited.  The 4D dataset for Movie 3a was edited to show only the cisternae marked by the arrow and arrowhead.  The cisterna marked by the arrow in Movie 3a is visible slightly earlier in this edited movie, because in Movie 3a that cisterna was initially obscured by another cisterna from a different focal plane.Movie S1a:  Animation of a cisternal maturation mechanism for Golgi transport.  Black dots represent a secretory cargo protein.  The green color represents an early Golgi resident protein, whereas the red color represents a late Golgi resident protein.  See Fig. S1a for further details. Movie S1b:  Animation of a stable compartments mechanism for Golgi transport.  The color scheme is as in Movie S1a.  See Fig. S1b for further details.Movie S2:  Example of a cisterna being tracked in a 4D dataset.  Shown are 20 consecutive z-stacks from Movie 1a, in which the cisternae were labeled with Sec7p‑GFP.   The arrows seen in two to four optical sections from each z ‑stack point to the same cisterna that is marked by the yellow arrow in Movie 1a.  This cisterna can be unambiguously tracked from one z‑stack to the next, and is clearly resolved from the other cisternae at each time point.Movie S3a:  GFP-Vrg4p labeling.  Shown are projected images of Golgi cisternae labeled with GFP‑Vrg4p.  The z‑stacks were taken at 1.5 s intervals.  The arrowhead and arrows mark three Golgi cisternae that acquired and then lost fluorescence.  This movie is labeled “B” in Table S1. Movie S3b:  GFP-Vrg4p labeling, edited.  The 4D dataset for Movie S3a was edited to show only the cisternae marked by the arrowhead and arrows. 

Losev et al. (2006) Nature 441, 1002-1006 Golgi maturation

visualized in living yeast

tagging the early Golgi with GFP–Vrg4 (GDP-mannose carrier) and the late Golgi with Sec7–DsRed

Movie 3a: nature04717-s13.mov

3b: nature04717-s14.mov

S1a: nature04717-s15.mov

Movie 4: nature04737-s07.mov

5: nature04737-s08.mov

6: nature04737-s09.mov

7: nature04737-s10.mov

Nature 441, 1007-1010, 2006 Kumi Matsuura-Tokita, Masaki Takeuchi, Akira Ichihara,

Kenta Mikuriya and Akihiko Nakano

Gos1 (green, medial) & Sec7 (Red, trans)

Deconvo-luted

mRFP-labelled Gos1 (medial-Golgi marker) and GFP-labelled Sec7 (trans marker). 色が遷移していく－

22: 471–478, 2010

Figure 3. 3D live imaging of a yeast Golgi cisterna. Current Opinion in Cell Biology 22: 471–478, 2010

Figure 4. Reticular connection between plant Golgi stacks.

左： Processing domain involves glycero-phospholipids (GPL).

右： Export Domain involves cholesterol and glycosphingolipids (SL).

ARF1 and SAR1 GTPases in Endomembrane Trafficking in PlantsInt J Mol Sci. Sep 2013; 14(9): 18181–18199.

脂質も differential sorting

Vesicle budding & fusion の道具箱1. Coatmer: budding の曲面を作る2. GTPases: Rab, Sar1, Arf3. SNARE: fusion machine （生得的に

intrinsically unfolded proteins ）(and 5. related coiled-coil and other

tethering factors etc)　　　４ . Dynamin GTPases pinching off the membrane and organelles ( これはかなり　　　　後に ) 　（ COPI, COPII には関与せず！？）

これらすべての総合・協力で可能になる

中野研究室最新結果Contact of cis-Golgi with ER exit sites executes cargo capture and delivery from the ER.Kazuo Kurokawa, Michiyo Okamoto & Akihiko NakanoNature Communications 5, Article number: 3653, 2014

ER exit site と (cis)Golgi の関係

Figure 1: cis-Golgi shows approach-and-contact actions towards the ERES.

Figure 2: cis-Golgi is stuck at the ERES when secretion is compromised.

Figure 4: cis-Golgi is stuck at the ERES when Uso1 function is impaired.

Figure 5: Cargo is transiently loaded into the ERES.

Figure 6: cis-Golgi captures cargo at the ERES.

Figure 7: A model of ER-Golgi cargo transport by a ‘hug-and-kiss’ action of cis-Golgi.

Supplementary Movie 1 (1,183 KB) 3D time-lapse observation of wild-type cells expressing Sec13-GFP (COPII coat protein, green) and mRFP-Sed5 (cis-Golgi, red). 2 x real time. White and red arrows indicate decreases (white) and increases (red) in the Sec13-GFP signal after associations between cis-Golgi and the ERES.

Supplementary Movie 2 (1,207 KB) 3D time-lapse observation of wild-type cells expressing Sec13-GFP (COPII coat protein, green) and Sec7-mRFP (trans-Golgi, red). 2 x real time. White and red arrows indicate decreases (white) and increases (red) in the Sec13-GFP signal after associations between trans-Golgi and the ERES.

Supplementary Movie 3 (1,006 KB) 3D time-lapse observation of sec3l-l cells expressing Axl2-GFP (cargo protein, green) and Mnn9-mCherry (cis-Golgi, red). 3.5 x real time.

Supplementary Movie 4 (3,120 KB) Another example of 3D movie showing sec3l-l cells expressing Axl2-GFP (cargo protein, green) and Mnn9-mCherry (cis-Golgi, red). 3.5 x real time.

Supplementary Movie 5 (12,385 KB) Magnified and rotated 3D movie of Axl2-GFP (cargo protein, green) and Mnn9-mCherry (cis-Golgi, red) showed in Supplementary movie 4.

Secretory pathway１． Coat

Structural & Evolutionary aspects

NCBI Books: Development

2. Coat GTPases: Sar1 and Arfs

2. RabGTPase

Vesicle flowの総元締め

Huge number of effectors!

Rab のサイクリカルな活性化機構とトランスロケーション  Rab は , GEP (=GEF), GDI, GAP の少なくとも３個の活性制御タンパク質により，その活性と局在がサイクリカルに制御されて機能している . GEP: GDP/GTP exchange protein, GDI: GDP dissociation inhibitor, GAP: GTPase-activating protein

Rab GTPases as coordinators of vesicle traffic Harald StenmarkNature Reviews Molecular Cell Biology 10, 513-525 (August 2009)

Rab66 種

酵母の 11 種の Rab とそのGAP/GEF 、 Effectros

Rabとその他のファクター

Rab ：すべての局面に関わるmaster regulator??

COPII bud

Rab1

p115

SNARE

TRAPP (transport protein particle) tether

Tethering to ERGICFinal fusion

Actin/MT motor

Rab domains

• Vesicle ではなく， target側にも

2, 107-117 (2001)

GEF->Rab->Effectors 　自身を集め他の Rab を寄せ付けないあるいは、他の Rab を呼び寄せる

3. SNARE

Molecular Biology of the Cell. 4th edition.

インフルエンザウィルスのような膜（宿主細胞由来）を持つウィルスの侵入は SNARE 類似の分子による

膜融合

Homotypic fusion の場合の SNARE

SNARE36 種

SNARE recruitment

Central ionic layer (red) and 15 hydrophobic layers (black)

Unfoldedからcoiled coilへ（安定化エネルギー）

• v-SNARE R-SNARE• t-SNARE Qa, Qb, Qc-SNARE (Gln/Asp)

SNAREpins 　　　～４ or more(?)/fusion

Variable N-term + SNARE domain + w/wo

Membrane helix*

SNARE complex formation

* 膜タンパク質だけれど， C- 末端にシグナルがあるので， post-translational targeting で膜に組み込まれる

J Cell Science 118, 3819-3828 (2005)

tt

v

Coiled-coil が膜を接着させ、融合させる

水を排除

Molecular Biology of the Cell. 4th edition.

Molecular Biology of the Cell. 4th edition.

Coiled-coil部分のTyr残基と脂質分子の相互作用もあり

Coiled-coil 形成前の　SNARE motif部分は、 unstructured(=unfolded) である。高次構造形成によって得られた自由エネルギー変化を膜融合に利用する。

膜貫通領域と Coiled-coil領域は共同して力を及ぼす

完全に融合した後の構造

cis-SNARE complex

SNARE-pin が膜貫通部分まで延び

さらに安定化！！

35 kBT/SNARE unit

膜融合に必要なエネルギーは 50-100 kBT

動物酵母でも 21種

Specificity factorSNARE36 種

沢山のSNARE標的膜を決める一つの仕組

み

蛋白質の役目は引き金で，実際に融合するのは脂質二重膜Cone and inverted cone

lipid molecules involved!

脂質分子も関係曲率の大きい膜に適した Minor lipid が合成・局在極性基と疎水性部分の大きさの違い・極性基の構造

（くさび形 /逆くさび形・ PI のリン酸化）

脱アシル化、アシル化でくさび形と逆くさび形の分子を作る

Hemifusion から Pore 形成で突然カーブが変わる

後始末N-Ethylmaleimide-sensitive Factor (NSF) 　（リング状

chaperone AAA-ATPase の１種）と

soluble NSF receptor (SNAP)

そもそも NSF→SNAP→SNARE=SNAP receptor の順に発見された

SNAP

NSF: AAA-ATPase の 1 種 もつれをほどく！

AAA (ATPases associated with diverse cellular activities) ほとんどこじつけですが .

http://mukb.medic.kumamoto-u.ac.jp/AAA/aaainfo.html

シナプスでは

Dynamin

複数の中間状態？

In vitro 系 SNARE だけでは一般に遅い

Ca2+ の効果の仕組みが今ひとつ分からない

JCB, Volume 172, Number 2, 281-293， 2006

Synaptotagmin as a Ca2+ sensor

Fusion detected by FRET

Fluoresence resonance energy transfer

Synaptotagmin’s cytosolic fragment enhances fusion event!

5. Long coiled-coil as a tether

SNARE に加え、それとは異なるcoiled-coil が小胞をつなぎとめる

Tethering

Long coiled-coil proteins

Rab, SNARE 以外の特異性決定因子

これ以外にも特異性を決める5. 可溶性巨大複合体 (10 種ほどのタンパク〜 1　MDa ）

Rab の活性化？？

Vesicle fusionSpecificity factors

• GTPases: >60 Rabs

• >30 SNARES 

• Long coiled-coil and other tethering complexes

Targeting － Tethering － Priming － Fusion

Organelle Identity

Cargo and cargo receptors*

Lipids (PIPx)*

Coat (G-proteins/Adaptor) Budding

Rab General Commander

Tether (coiled coil/multimeric) Tethering

SNARE* 　　　　　　　　　　　　 Fusion

*These are mixed up after fusion events.

Coat は３種類何が特異性を決めているか？？Cargo に対してはSar1, Arf + Adapter膜特異性ではSNARE, TetherRab and its effectors(>60 Rabs and >30 SNAREs)

Posttranslational modification of proteins

Extracellular domains• Sugar chain: ER to Golgi• GPI-anchor: ERCytosolic• Phosphorylation• Ubiquitination, SUMOylation• Isoprenylation: Rab Membrane targeting• Fatty acylation (palmitoyl, myristoyl): Arf　　 Lipid-Linked Proteins

膜貫通 αヘリックスを持たないPeripheral or extrinsic 膜タンパク質

の膜への結合

GPI-anchored proteinsプリオン　 LPL

別の図

GPI-linked proteins －　 Lipoprotein Lipase

Prion protein

Signal sequence＋分泌タンパク＋ GPI 結合サイト＋膜貫通領域

GPI linker 合成も複雑だが、転移酵素も複

雑

N-Linked sugar chains

Carrier-Maturation/Progression Model

Fusion of retrograde and anterograde vesicles forming the cis-membrane (with high fusogenic abillity) b1

Formation of connection between cis and medial cisternae b2

Redistribution of membranes within Golgi together with maturation b3

Fusion of trans-Golgi/TGN b4 Golgi Structure Video

隣同士の連結もある

Mitosis の時には Golgiは一旦消滅すぐ再生

Cyclin による GM130 のリン酸化（左）とタンパク質の分布（右）

Golgi

ER

動物細胞

Direct fragmentation model

Golgi の小胞化

ER recycling model

ER に吸収される

カビ毒の Brefeldin

Arf を阻害するので COPI ができない

それでなぜ、 Golgi がなくなるか？

通常は COPI に集まる SNARE が広く分布して、他の膜 ER などと融合

さて Golgi 以降Protein sorting in the trans Golgi network

行き先いくつか

• Lysosome

• Cell membrane

=Secretory vesicles

(Regulated & Constitutive)

プラス• Retrograde traffic: 細胞膜その他から

Golgi, ER へ

Lysosomes Acidic compartments containing acid hydrolases

H+ pumping through V-type ATPase

植物細胞では

植物細胞は水ぶくれ

A large increase in cell volume can be achieved without increasing the volume of the cytosol.

Strands of cytosol are stabilized by bundles of actin filaments.

細胞の分解工場

消化の材料は　細胞の外からと　細胞内

Endosome と Autophagosome

老化と関連する

Autophagyむしろ寿命を延長する

最近では新生児で起こるとの報告あり

Lysosomal Transport

Mannose-P が Lysosome 行きシグナル