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  1. 020 学位論文
  2. 高エネルギー加速器科学研究科
  3. 13 物質構造科学専攻

Structural and biochemical studies on protein complexes regulating membrane traffic

https://ir.soken.ac.jp/records/4063
https://ir.soken.ac.jp/records/4063
55fb7f23-6223-46fa-bbdc-75c04d05449a
名前 / ファイル ライセンス アクション
甲1586_要旨.pdf 要旨・審査要旨 (325.0 kB)
Item type 学位論文 / Thesis or Dissertation(1)
公開日 2013-11-15
タイトル
タイトル Structural and biochemical studies on protein complexes regulating membrane traffic
タイトル
タイトル Structural and biochemical studies on protein complexes regulating membrane traffic
言語 en
言語
言語 eng
資源タイプ
資源タイプ識別子 http://purl.org/coar/resource_type/c_46ec
資源タイプ thesis
著者名 中村, 健介

× 中村, 健介

中村, 健介

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フリガナ ナカムラ, ケンスケ

× ナカムラ, ケンスケ

ナカムラ, ケンスケ

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著者 NAKAMURA, Kensuke

× NAKAMURA, Kensuke

en NAKAMURA, Kensuke

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学位授与機関
学位授与機関名 総合研究大学院大学
学位名
学位名 博士(理学)
学位記番号
内容記述タイプ Other
内容記述 総研大甲第1586号
研究科
値 高エネルギー加速器科学研究科
専攻
値 13 物質構造科学専攻
学位授与年月日
学位授与年月日 2013-03-22
学位授与年度
値 2012
要旨
内容記述タイプ Other
内容記述 In order to gain insights into regulations of various membrane structures in cell, structural
and functional studies were carried out on two target proteins: Arfaptin, a protein
involved in tubule formation at trans-Golgi network; and Atg16L, a protein involved
in formation of autophagosomes.
The BAR (Bin/Amphiphysin/Rvs) domain forms a curved helical homodimer that can
sense or induce the curvature of the membrane it associate with. BAR domain binds to acidic
membranes with its basic concaved face in a rather nonspecific manner. This is in contrast to
other membrane-binding domains, many of which recognize the head groups of specific
phospholipids. BAR domains are often found in conjunction with other membrane-binding
modules, such as a PH (pleckstrin homology) domain and a PX (phox homology) domain,
which define spatial and functional specificities in membrane association. For instance, many
members of the Sorting Nexin (SNX) family, including SNX9, have two membrane-binding
properties attributable to its curvature sensing BAR domain and phosphoinositide-binding PX
domain; this allows for the selective targeting to high curvature sub-domains of endosomal
compartments enriched in specific phosphopnositides. Upon associating with target
membranes, the SNX PX-BAR domain drives membrane tubulation for tubule-based sorting.
BAR domain-containing protein Arfaptin functions in both tubule formation and
stabilization; Arfaptin-1 stabilizes the fission sites of secretory granules at the trans-Golgi
network; Arfaptin-2 induces the membrane tubules in Golgi-membranes. The two isoforms
have amino acid sequence similarity of 68% and share similar domain structures. Both
isoforms localize in the Golgi region, when exogenously expressed in cells, through the
association with small GTPase Arf-like 1 (Arl1). Arfaptin-2 colocalizes with Arl1 on dynamic
vesicular and tubular structures emanating from the Golgi, suggesting that Arl1 regulates
Arfaptin-mediated membrane deformation at the trans-Golgi.
The two isoforms of Arfaptin are similarly affected by Arl1, but they associate with
different regulators. Arfaptin-2, but not Arfaptin-1, associates with another small GTPase
Rac1. The crystal structures of the Rac1–Arfaptin-2 BAR domain complex, reported by
Tarricone, et al, show how Rac1 binds to the concaved face of the Arfaptin-2 BAR
homodimer, in the way that would interfere with the membrane-association of the BAR
domain. In addition, Rac1 also interferes with the Arf-association of Arfaptin-2. Hence, the
function of Arfaptin-2 is intricately regulated by the crosstalk between the small GTPases,
Arf/Arl1 and Rac1. However, the molecular basis of this crosstalk remained unrevealed, for
the complex structure of Arl1–Arfaptin-2 was not known.
The crystal structures were determined for Arfaptin-1 in free form and Arfaptin-1 or
Arfaptin-2 in complex with Arl1. In the complex structures, two molecules of Arl1 were
symmetrically bound on each side of the crescent-shaped homodimer of Arfaptin BAR
domain, leaving the concave face open for membrane association. This conformation provided
the structural basis for recruitment of Arfaptins onto Golgi membranes by Arl1. Arl1 and
Rac1, another binding partner of Arfaptin-2, bound to Arfaptin-2 with the mM-order
dissociation constants in Surface Plasmon Resonance (SPR) experiments. Structural
comparison between Rac1–Arfaptin-2 and Arl1–Arfaptin-2, combined with SPR experiments,
indicated that Rac1 interferes with the one of two molecules of Arl1 on Arfaptin-2. These
results provided structural basis for the recruitment of Arfaptins onto Golgi membranes by
Arl1 and the crosstalk between Arf/Arl1 and Rac1.
Autophagy is the basic catabolic process in Eukaryotic cells. The process involves unique
trafficking event in which cytoplasmic constituents are isolated and delivered from the
cytoplasm to lysosome for degradation. In addition to the basic roles in catabolism, the
pathway is also utilized in the elimination of intracellular pathogen and presentation of antigen
in Mammal. There are three different types of autophagy: chaperone-mediated autophagy,
microautophagy, and macroautophagy. In the chaperone-mediated autophagy, degradation
targets that contain specific motif are recognized and translocated into lysosomes by
chaperone heat shock cognate 70 (Hsc70) and the receptor lysosome-associated membrane
protein 2A (LAMP2A). In the microautophagy, lysosome directly engulfs the cytoplasmic
components. In the macroautophagy, cellular constituents are isolated by the transient
double-membrane–bound structure called autophagosome and delivered to lysosome by the
fusion of the two membrane-bound structures.
The autophagosomes originate from a small single-layer membrane structure termed
Precursor Membrane Structure (PAS). Through hemi-fusions with additional membrane
structures, PAS develops into a flat double-membrane structure, termed the isolation
membrane, which expands and enwraps portions of the cytoplasm. As their ends meet, it
results in a large double-membrane vesicle, or an autophagosome, that isolates the degradation
targets. The maturation of autophagosome involves an ubiquitin-like conjugation system in
Autophagy-related protein (Atg) family; in which LC3, a mammalian homologue of yeast
Atg8, is conjugated to phosphatidylethanolamine (PE) by E3-like ligase Atg16(L) complex.
Ethanolamine-conjugated LC3 and its orthologue GATE-16, as well as the yeast Atg8,
promote tethering and hemi-fusion of membranes in vitro. Thus the conjugation system is
considered essential for the autophagosome maturation. Atg16L complex is suggested to
function during the elongation step, as the complex dissociates from the membrane upon the
completion of autophagosome.
The structure and function of those Atg proteins are extensively studied in yeast. Multiple
crystal structures are determined for the fragments of the yeast Atg16 complex, which consists
of Atg5-Atg12 conjugate and Atg16. The model of the overall structure has been proposed by
Fujioka, et al; Atg5-Atg12 conjugate binds to N-terminal helix of Atg16, which dimerizes
through its C-terminal coiled-coil domain to form a complex that contains two copies of each
of the three Atg proteins. The complex associates with membranes through Atg5, and induce
homotypic tethering of vesicles in vitro even in the absence of Atg8. It has confirmed in vivo
and in vitro that Atg16 is required for the efficient conjugation of LC3 and PE. Moreover, the
dimerization of Atg16 at the coiled-coil domain is reported to be essential for autophagy in
vivo.
The corresponding protein complex in Mammal consists of Atg5-Atg12 conjugate and
Atg16L; thus named Atg16L complex. The mammalian Atg16L complex is similar to the
yeast Atg16 complex: It associates with membranes through Atg5; dimerizes through the
coiled-coil domain of Atg16L; and has E3-ligase activity toward LC3, the homologue of Atg8.
However, Atg16L is significantly different from the yeast Atg16. It binds to small GTPase
Rab33 at the region following the coiled-coil domain, and also has an additional WD40
domain the C-terminus. Recent reports identified a single nucleotide polymorphism in
N-terminus of the WD40 domain that is associated with inflammatory Crohn's disease. Thus,
those additional regions of Atg16L may be involved in the distinct mammalian-autophagic
functions that are absent in yeast.
The mammalian Atg16L directly interacts with Rab33b, the Golgi-resident small GTPase
involved in Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking. Rab33b
interacts with Atg16L, at the region following the coiled-coil region (residues 80-200), and
recruits not only Atg16L but also Atg12, which does not directly bind Rab33b. This indicates
that Rab33b is able to recruit the entire Atg16L complex. Indeed, the expression of
Rab33b-binding domain of Atg16L strongly inhibited autophagosome formation. However,
the molecular mechanism of this recruitment and subsequent function of Atg16L complex
remained unrevealed, for the structure of the mammalian Atg16L and its complex with
Atg5-Atg12 is not yet known.
The X-ray crystal structure was determined for the coiled-coil domain of mouse Atg16L;
which had an antiparallel coiled-coil dimer in contrast to the yeast parallel Atg16. The
dimerization interface had hydrophobic interactions and hydrogen bonds at the residues that
were not conserved between mammal and yeast. These findings suggested that the overall
structure of mammalian Atg16L complex is quite distinct form that of the yeast Atg16
complex.
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