MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
批准号:
2179653
负责人:
Gregory S Payne
金额:
$26.04万
依托单位国家:
美国
项目类别:
财政年份:
1988
资助国家:
美国
项目状态:
已结题
起止时间:
1988-02-01 至 1997-01-31
关键词:
Golgi apparatus Saccharomyces cerevisiae cell growth regulation clathrin density gradient ultracentrifugation endoplasmic reticulum fungal genetics gel electrophoresis gene expression laboratory rabbit membrane proteins molecular cloning mutant nucleic acid sequence pheromone pinocytosis protein transport receptor mediated endocytosis temperature sensitive mutant western blottings
中文摘要
真核细胞的区室组织依赖于精确的
蛋白质分布到不同的隔室和保留
每个隔室中的常驻蛋白质。 蛋白质的分类能力
具有不同目的地是运输机械的关键特征
其控制蛋白质分布和保留。 的分子基础
这种选择性是我们建议的重点。
在网格蛋白重链中携带突变(chc 1)的酵母菌株的研究
链亚基揭示了网格蛋白在几个
选择性蛋白质转运步骤,包括选择性保留高尔基体
膜蛋白,受体介导的交配肽内吞作用
α-因子,和蛋白质从高尔基体的排序,
溶酶体样空泡。 基于网格蛋白在高尔基体膜上的功能
蛋白质保留,采用遗传筛选来分离两个
高尔基体膜缺陷的其它突变体(lam突变体)
蛋白质滞留 这些研究提供了一个综合的基础
遗传学和生物化学方法来研究三个方面,
选择性蛋白质转运:1)高尔基体膜蛋白的机制
保留; 2)网格蛋白相关蛋白在每个细胞中所起的作用
网格蛋白依赖的转运过程; 3)网格蛋白-
介导的α-因子内吞作用。
网格蛋白与高尔基体膜蛋白的相互作用将被研究
通过网格蛋白包被的囊泡的分级分离进行生物化学,和
通过确定突变体高尔基体膜的定位,
在CHC1突变体中缺乏胞质滞留信号的蛋白质。
另外的高尔基体膜蛋白保留缺陷的lam突变体将
鉴定并用于克隆LAM基因的野生型版本。
针对LAM基因产物产生的抗体将用于表征
Lam蛋白 为了进一步确定网格蛋白涂层在
选择性转运,网格蛋白相关蛋白(AP)编码基因
将被克隆并用于产生基因破坏。 网格蛋白依赖性
将在突变株中监测转运途径。 不同的AP和
将结合chc1突变来探索遗传相互关系。
生化分级将用于评估物理相互作用
不同AP之间以及AP和网格蛋白之间的差异。 最后,体外
网格蛋白依赖性选择性转运的测定将由
在透化细胞中重建α-因子内吞作用。 的
将鉴定体外反应的功能组分,
特征在于透化突变细胞的互补,或
细胞质的分级分离。 总之,这些研究将确定
以前未识别的选择性蛋白质转运组分
机制,并解决每个确定的机构发挥的具体作用,
参与者。
英文摘要
The compartmental organization of eukaryotic cells relies on accurate
distribution of proteins to different compartments and on retention of
resident proteins within each compartment. The ability to sort proteins
with different destinations is a key feature of the transport machinery
which governs protein distribution and retention. The molecular basis
of this selectivity is the focus of our proposal.
Studies of yeast strains carrying mutations (chc1) in the clathrin heavy
chain subunit revealed that clathrin plays an important role in several
selective protein transport steps, including selective retention of Golgi
membrane proteins, receptor-mediated endocytosis of the mating peptide
alpha-factor, and sorting of proteins from the Golgi apparatus to the
lysosome-like vacuole. Based on clathrin's function in Golgi membrane
protein retention, a genetic screen was employed to isolate two
additional mutants (lam mutants) which are defective in Golgi membrane
protein retention. These studies provide the basis for a combined
genetic and biochemical approach to investigate three aspects of
selective protein transport: 1) the mechanism of Golgi membrane protein
retention; 2) the role played by clathrin-associated proteins in each
clathrin-dependent transport process; 3) the mechanism of clathrin-
mediated alpha-factor endocytosis.
Clathrin's interaction with Golgi membrane proteins will be investigated
biochemically by fractionation of clathrin-coated vesicles, and
genetically by determining the localization of mutant Golgi membrane
proteins lacking cytoplasmic retention signals in chc1 mutants.
Additional lam mutants defective in Golgi membrane protein retention will
be identified and used to clone wild-type versions of LAM genes.
Antibodies raised against LAM gene products will be used to characterize
the Lam proteins. To further define the function of clathrin coats in
selective transport, genes encoding clathrin-associated proteins (APs)
will be cloned and used to generate gene disruptions. Clathrin-dependent
transport pathways will be monitored in mutant strains. Different ap and
chc1 mutations will be combined to explore genetic interrelationships.
Biochemical fractionations will be used to assess physical interactions
between different APs and between APs and clathrin. Finally, an in vitro
assay for clathrin-dependent selective transport will be developed by
reconstituting alpha-factor endocytosis in permeabilized cells. The
functional components of the in vitro reaction will be identified and
characterized by complementation of permeabilized mutant cells or
fractionation of cytosol. Together, these studies will identify
previously unrecognized components of the selective protein transport
machinery and address the specific roles played by each identified
participant.
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会议论文
Molecular studies of selective protein transport
-
批准号:7891051
-
项目类别:
-
资助金额:$32.22万
-
财政年份:2009
-
负责人:Gregory S Payne
-
依托单位:
SYSTEMATIC IDENTIFICATION AND CLASSIFICATION OF UBIQUITIN-BINDING MOTIFS IN SAC
-
批准号:7182438
-
项目类别:
-
资助金额:$0.72万
-
财政年份:2005
-
负责人:Gregory S Payne
-
依托单位:
Clathrin adaptor function at the TGN and endosomes
-
批准号:6876069
-
项目类别:
-
资助金额:$27.17万
-
财政年份:2004
-
负责人:Gregory S Payne
-
依托单位:
CLATHRIN COATED VESICLE INTERACTING PROTEINS
-
批准号:6979564
-
项目类别:
-
资助金额:$0.34万
-
财政年份:2004
-
负责人:Gregory S Payne
-
依托单位:
Clathrin adaptor function at the TGN and endosomes
-
批准号:7048606
-
项目类别:
-
资助金额:$26.57万
-
财政年份:2004
-
负责人:Gregory S Payne
-
依托单位:
Clathrin adaptor function at the TGN and endosomes
-
批准号:6773638
-
项目类别:
-
资助金额:$27.0万
-
财政年份:2004
-
负责人:Gregory S Payne
-
依托单位:
Clathrin adaptor function at the trans Golgi network and endosomes
-
批准号:7214809
-
项目类别:
-
资助金额:$25.8万
-
财政年份:2004
-
负责人:Gregory S Payne
-
依托单位:
IDENTIFICATION/CLASSIFICATION OF UBIQUITIN-BINDING MOTIF
-
批准号:6979558
-
项目类别:
-
资助金额:$0.34万
-
财政年份:2004
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
-
批准号:6151044
-
项目类别:
-
资助金额:$29.11万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
-
批准号:2179655
-
项目类别:
-
资助金额:$28.29万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
Molecular studies of selective protein transport
-
批准号:8212359
-
项目类别:
-
资助金额:$36.98万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
-
批准号:2022179
-
项目类别:
-
资助金额:$26.29万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
-
批准号:2654949
-
项目类别:
-
资助金额:$27.28万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF CLATHRIN-COATED MEMBRANE FUNCTION
-
批准号:3295822
-
项目类别:
-
资助金额:$10.7万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF CLATHRIN-COATED MEMBRANE FUNCTION
-
批准号:3295824
-
项目类别:
-
资助金额:$12.88万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
-
批准号:6498662
-
项目类别:
-
资助金额:$35.08万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
Molecular Studies of Selective Protein Transport
-
批准号:8816106
-
项目类别:
-
资助金额:$37.15万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT
-
批准号:2872657
-
项目类别:
-
资助金额:$28.2万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
MOLECULAR STUDIES OF CLATHRIN-COATED MEMBRANE FUNCTION
-
批准号:3295826
-
项目类别:
-
资助金额:$16.76万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
Molecular Studies of Selective Protein Transport
-
批准号:7009579
-
项目类别:
-
资助金额:$36.65万
-
财政年份:1988
-
负责人:Gregory S Payne
-
依托单位:
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