细胞学第9篇B.pptVIP

?信号序列(signal sequence) ◆研究过程 ●1960s ,George Palade 示踪技术研究培养细胞中蛋白质的分泌 ●Redman和Sabatini用分离的RER小泡研究膜结合核糖体合成的蛋白质去向 ●1971年美国洛克菲勒大学的Blobel等提出了信号序列(signal sequence)的概念。 ◆信号序列的间接证据 1972年, Milstein 和他的同事发现: ●从细胞质分离的IgG轻链: 多20个氨基酸残基 ●释放到细胞外的IgG轻链: 少20个氨基酸残基 ◆信号肽的证实 G.Blobel等用离体实验证实了信号肽的存在: ●RER对产物的影响 ●蛋白水解酶实验 ●多聚核糖体的离体翻译 ?信号肽的特性 ◆序列特征 15-35个氨基酸残基，其中含有4-12个疏水残基 ◆特异性: ◆位置: ●N-端突出的一段肽 ●内含信号肽。 ?早期信号假说(signal hypothesis) 1975年，Blobel等正式提出了信号假说,要点是: ◆合成始于游离核糖体; ◆N端信号序列靠自由碰撞与内质网膜接触,然后插入内质网的膜; ◆蛋白质以袢环形式穿过内质网的膜; ◆蛋白的存在方式: ●若信号序列被信号肽酶切除:完全进入腔 ●若是停止转移信号: 膜蛋白? ?信号识别颗粒与停靠蛋白 ◆signal recognition partical, SRP ●SRP is a cytosolic ribonucleoprotein particle ● Six discrete polypeptides ● A 300-nucleotide RNA ●三个功能域: ▲翻译暂停结构域(P9/P14) ▲信号肽识别结合位点(P54) ▲SRP受体蛋白结合位点(P68/P72)。 ◆Docking protein, DP ●是SRP在内质网膜上的受体蛋白 ●两个亚基:亲水的α亚基, 疏水的β亚基 ◆易位子( translocon) ●Multiple copies of the Sec61 complex assemble to form a cylinder translocon channel ●5～6 nm high and 8.5 nm in diameter, with a central pore, roughly 2 nm in diameter ●哺乳动物细胞中有三种类型的Sec61, 即α、β和γ ●Sec61α有10个跨膜α螺旋 Orientation of menbrane proteins ?跨膜信号与膜蛋白的定向 ◆单次跨膜蛋白 ? ●起始转移信号(start transfer signal) ●终止转移序列(stop transfer sequence) ●可切割的信号序列(Cleavable signal sequence) (N-端) ●内部信号序列(internal signal sequence) (internal noncleavable signal sequences ) ◆单次跨膜蛋白的定向 ●Cleavable signal sequence amino terminus exposed to the cytosol ●Internal signal sequence ▲Amino terminus exposed to the cytosol ▲Carboxy terminus exposed to the cytosol ◆停止转运信号与多次跨膜蛋白的定向 ●产生: Proteins that span the membrane multiple times are thought to be inserted as a result of an alternating series of internal signal sequences and transmembrane stop-transfer sequences. ●定向:? ?Protein Folding and Processing in the ER ◆Protein folding in the ER ●Bip蛋白∶ Bip是一类分子伴侣是重链结合蛋白的简称(heavy-chain binding protein) ●BiP is thought to bind to the unfolded polypeptide chain as it crosses the membrane and then mediates protein folding and the assembly of multisu