membrane is required to make them. In liver and pancreatic cells, for example, the endoplasmic reticulum has a total membrane surface area that is, respectively, 25 times and 12 times that of the plasma membrane (Table 12-2). The membraneenclosed organelles are packed tightly in the cytoplasm, and, in terms of area and mass, the plasma membrane is only a minor membrane in most eukaryotic cells (Figure 12-2).
膜是制造它们所必需的。例如，在肝脏和胰腺细胞中，内质网的总膜表面积分别是质膜的 25 倍和 12 倍（表 12-2）。膜包围的细胞器在细胞质中紧密堆积，从面积和质量来看，质膜在大多数真核细胞中仅占少量膜（图 12-2）。

Evolutionary Origins Explain the Topological
Relationships of Organelles
进化起源解释了细胞器的拓扑关系

Second, the ancestral plasma membrane that surrounded the genome is now an internal membrane that becomes the inner nuclear membrane. Because of how it originated, the inner nuclear membrane is continuous with other plasma membrane-derived internal membranes, including the outer nuclear membrane. Specialized structures, the nuclear pore complexes, are located at points where the inner and outer nuclear membranes connect and provide a conduit for communication between the nucleus and cytosol. Segregation of an
其次，围绕基因组的祖先质膜现在成为了内膜，形成内核膜。由于其起源，内核膜与其他来源于质膜的内膜是连续的，包括外核膜。特殊结构——核孔复合体，位于内核膜和外核膜连接的地方，为核与细胞质之间的交流提供了通道。

Third, symbionts that were originally outside the cell were trapped inside the cell and became endosymbionts. At some point, endosymbionts escaped from their membrane enclosure into the cytosol where they eventually became mitochondria and plastids that contain their own genomes. The nature of these genomes and the close resemblance of the proteins in these organelles to those in some present-day bacteria provide strong evidence for their endosymbiont origins (see Figure 14-55). Like the bacteria from which they were derived,
第三，最初位于细胞外的共生体被困在细胞内，成为内共生体。在某个时刻，内共生体从其膜包围中逃逸到细胞质中，最终演变为含有自身基因组的线粒体和质体。这些基因组的性质以及这些细胞器中蛋白质与某些现代细菌中蛋白质的高度相似性为它们的内共生体起源提供了有力证据（见图 14-55）。与其来源的细菌一样，

(A)

(B)

Macromolecules Can Be Segregated Without a Surrounding Membrane
大分子可以在没有周围膜的情况下被分离

Biomolecular condensates can be found in all organisms (Figure 12-6), and eukaryotic cells contain a dozen or more different types (Table 12-3). The sizes of the known condensates range from $\sim 50\mathrm{nm}$$\sim 50\mathrm{nm}$∼50nm\sim 50 \mathrm{~nm} in diameter (slightly bigger than ribosomes) to a micrometer or more in the case of nucleoli. These structures include many different types of ribonucleoprotein condensates (some in the nucleus and
生物分子凝聚体可以在所有生物中找到（图 12-6），真核细胞包含十种或更多不同类型（表 12-3）。已知凝聚体的大小范围从 $\sim 50\mathrm{nm}$$\sim 50\mathrm{nm}$∼50nm\sim 50 \mathrm{~nm} 直径（略大于核糖体）到在核仁的情况下达到一个微米或更大。这些结构包括许多不同类型的核糖核蛋白凝聚体（一些位于细胞核中）。

Figure 12-5 Biomolecular condensates formed by scaffold macromolecules recruit clients. As described in Chapter 3 (see Figure 3-77), a set of macromolecules that participate in weak, dynamic, and multivalent interactions (shown in red) with each other can form a biomolecular condensate (Movie 12.1). The macromolecules that directly participate in formation of the condensate are termed “scaffolds.” The scaffold proteins and RNA can recruit other macromolecules, termed “clients,” via specific interactions. Condensate formation does not depend on these clients, but they are part of the condensate because of their specific scaffold interactions.
图 12-5 由支架大分子形成的生物分子凝聚体招募客户。如第 3 章所述（见图 3-77），一组参与弱的、动态的和多价相互作用（以红色显示）的宏观分子可以形成生物分子凝聚体（电影 12.1）。直接参与凝聚体形成的宏观分子称为“支架”。支架蛋白和 RNA 可以通过特定相互作用招募其他宏观分子，称为“客户”。凝聚体的形成并不依赖于这些客户，但由于它们与支架的特定相互作用，它们是凝聚体的一部分。

Multivalent Interactions Mediate Formation of Biomolecular Condensates
多价相互作用介导生物分子凝聚体的形成

Biomolecular Condensates Create Biochemical Factories
生物分子凝聚体创造生化工厂

The fluctuating network of interactions among the macromolecules inside of a condensate excludes other macromolecules from the surrounding environment. By contrast, nucleotides, metabolites, cofactors, and other small
在冷凝物内部，大分子之间波动的相互作用网络排除了周围环境中的其他大分子。相比之下，核苷酸、代谢物、辅因子和其他小分子则可以自由进出。
(A)

(B)
$20\mu \mathrm{m}$$20\mu \mathrm{m}$20 mum20 \mu \mathrm{~m}
Figure 12-7 Condensates with different properties can coexist as part of a larger structure. The nucleolus is a condensate that is composed of three morphologically and functionally distinct regions, one inside the other, each formed by a different set of scaffold macromolecules. (A) In the experiment shown, the scaffolds from two nucleolar substructures have been purified-fibrillarin from the nucleolus’s fibrillar component and nucleophosmin from its granular component. Both of these scaffold proteins contain binding sites for RNA, and when mixed with RNA in a test tube, each purified scaffold will assemble into an RNA-protein condensate, as illustrated at left. However, as illustrated at right, when mixed together they instead form a multilayered structure that has one type of condensate encased inside the other type of condensate. (B) The condensates, formed either separately or together, viewed by fluorescence microscopy; fibrillarin is green and nucleophosmin is red. (Adapted from M. Feric et al., Cell 165:1686-1697, 2016.)
图 12-7 具有不同性质的凝聚物可以作为更大结构的一部分共存。核仁是一个由三个形态上和功能上不同的区域组成的凝聚物，这些区域一个在另一个内部，每个区域由不同的支架大分子组成。(A) 在所示实验中，来自两个核仁亚结构的支架已被纯化——来自核仁纤维成分的纤维蛋白和来自其颗粒成分的核磷蛋白。这两种支架蛋白都含有 RNA 的结合位点，当与 RNA 在试管中混合时，每种纯化的支架将组装成 RNA-蛋白凝聚物，如左侧所示。然而，如右侧所示，当它们混合在一起时，反而形成了一种多层结构，其中一种类型的凝聚物被包裹在另一种类型的凝聚物内部。(B) 通过荧光显微镜观察到的凝聚物，无论是单独形成还是一起形成；纤维蛋白呈绿色，核磷蛋白呈红色。(改编自 M. Feric 等，Cell 165:1686-1697, 2016。)
molecules can rapidly diffuse into the condensate where they can engage with the enzymes that reside there. The product of these enzymes within the condensate can be used by other enzymes that are coresident in the condensate before the product diffuses away. In this way, multistep reactions can be accelerated to rates beyond that possible without co-segregation of the enzymes inside a condensate.
分子可以迅速扩散到冷凝物中，在那里它们可以与驻留的酶相互作用。这些酶在冷凝物内的产物可以被其他与其共存的酶使用，随后该产物再扩散出去。通过这种方式，多步反应的速率可以加快，超过在没有酶在冷凝物内共同分离的情况下所能达到的速率。

Consider for example, the pyrenoid, a complex structure found in many algae that contains a condensate enriched in the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and a pyrenoid-specific scaffolding protein. The Rubisco-containing condensate is interwoven with membrane tubules. Carbonic anhydrase inside the membrane tubule converts bicarbonate $\left({\mathrm{HCO}}_3^{-}\right)$$\left({\mathrm{HCO}}_3^{-}\right)$(HCO_(3)^(-))\left(\mathrm{HCO}_{3}^{-}\right)into ${\mathrm{CO}}_2$${\mathrm{CO}}_2$CO_(2)\mathrm{CO}_{2}, which Rubisco uses to carboxylate ribulose 1,5-bisphosphate (Figure 12-8). This carboxylation reaction is a critical early step in carbon fixation during photosynthesis (discussed in Chapter 14). If Rubisco were not in a condensate in close proximity to carbonic anhydrase, the low free ${\mathrm{CO}}_2$${\mathrm{CO}}_2$CO_(2)\mathrm{CO}_{2} concentration combined with a competition by oxygen for Rubisco’s active site would favor reaction with oxygen (termed “photorespiration”; see Chapter 14, p. 847) over carbon fixation. Land plants do not need a pyrenoid to fix carbon because
例如，考虑到焦体，这是一种在许多藻类中发现的复杂结构，包含富含酶核酮糖-1,5-二磷酸羧化酶/氧化酶（Rubisco）和特定于焦体的支架蛋白的浓缩物。含 Rubisco 的浓缩物与膜小管交织在一起。膜小管内的碳酸酐酶将碳酸氢盐 $\left({\mathrm{HCO}}_3^{-}\right)$$\left({\mathrm{HCO}}_3^{-}\right)$(HCO_(3)^(-))\left(\mathrm{HCO}_{3}^{-}\right) 转化为 ${\mathrm{CO}}_2$${\mathrm{CO}}_2$CO_(2)\mathrm{CO}_{2} ，Rubisco 利用该物质对核酮糖-1,5-二磷酸进行羧化（见图 12-8）。这一羧化反应是光合作用中碳固定的关键早期步骤（在第 14 章中讨论）。如果 Rubisco 不在与碳酸酐酶紧密相邻的浓缩物中，低自由 ${\mathrm{CO}}_2$${\mathrm{CO}}_2$CO_(2)\mathrm{CO}_{2} 浓度加上氧气对 Rubisco 活性位点的竞争将使反应更倾向于与氧气反应（称为“光呼吸”；见第 14 章，第 847 页），而不是碳固定。陆生植物不需要焦体来固定碳，因为

Biomolecular Condensates Form and Disassemble in Response to Need
生物分子凝聚体根据需求形成和解聚

For example, the condensates called stress granules only form during certain types of cellular stress, and they dissolve when the stress is alleviated. These condensates are enriched in translationally inactive mRNAs, various translation factors, ribosomal subunits, and various RNA-binding proteins. They
例如，称为应激颗粒的冷凝物仅在某些类型的细胞应激期间形成，并在应激缓解时溶解。这些冷凝物富含转录不活跃的 mRNA、各种翻译因子、核糖体亚基和各种 RNA 结合蛋白。

Proteins Can Move Between Compartments in Different Ways
蛋白质可以通过不同的方式在细胞区室之间移动

1. In protein translocation, transmembrane protein translocators directly transport specific proteins from the cytosol into a space that is topologically distinct: either the other side of a membrane or within the lipid bilayer in the case of integral membrane proteins. The transported protein molecule usually must unfold to snake through the translocator. The initial transport of selected proteins from the cytosol into the ER lumen, the ER membrane, or mitochondria occurs in this way.
   在蛋白质转运中，跨膜蛋白转运体直接将特定蛋白质从细胞质运输到一个拓扑上不同的空间：要么是膜的另一侧，要么是在整体膜蛋白的情况下位于脂质双层内。被转运的蛋白质分子通常必须展开以便穿过转运体。选定蛋白质从细胞质初步运输到内质网腔、内质网膜或线粒体就是以这种方式进行的。
2. In gated transport, proteins and RNA molecules move between the cytosol and the nucleus through nuclear pore complexes in the nuclear envelope. The nuclear pore complexes function as selective gates that support the active transport of specific macromolecules and macromolecular assemblies between the two topologically equivalent spaces.
   在门控运输中，蛋白质和 RNA 分子通过核膜中的核孔复合体在细胞质和细胞核之间移动。核孔复合体作为选择性通道，支持特定大分子和大分子组装在这两个拓扑等价空间之间的主动运输。
3. In vesicular transport, membrane-enclosed transport intermediateswhich may be small, spherical transport vesicles, elongated tubules, or larger, irregularly shaped fragments of organelles-ferry proteins from one topologically equivalent compartment to another. The transport intermediate becomes loaded with a cargo of molecules derived from the lumen and membrane of the originating compartment as it buds and pinches off. At the destination compartment, the transport intermediate fuses with the compartment’s enclosing membrane to discharge its cargo
   在囊泡运输中，膜封闭的运输中间体可以是小型的球形运输囊泡、延长的管状物或较大、不规则形状的细胞器碎片，它们将蛋白质从一个拓扑等效的腔室运输到另一个腔室。当运输中间体从起始腔室的腔道和膜中出芽并断裂时，它会装载来自这些区域的分子货物。在目的腔室，运输中间体与腔室的包膜融合，以释放其货物。

Figure 12-10 A simplified “road map” of protein traffic within a eukaryotic cell. Proteins can move from one compartment to another by protein translocation (blue), gated transport (red), vesicular transport (green), or engulfment (gray). The sorting signals that direct a given protein’s movement through the system, and thereby determine its eventual location in the cell, are contained in each protein’s amino acid sequence. The journey begins with the synthesis of a protein on a ribosome in the cytosol and, for many proteins, terminates when the protein reaches its final destination. Other proteins shuttle back and forth between the nucleus and cytosol. At each intermediate station (boxes), a decision is made as to whether the protein is to be retained in that compartment or transported further. A sorting signal may direct either retention in or exit from a compartment. A special transport process termed “engulfment” is used to move proteins from the cytosol into the lysosome in autophagy or used to enclose chromosomes inside the nucleus during nuclear envelope re-formation after mitosis. The movement of macromolecules into and out of a condensate is not shown here. This process does not involve crossing a membrane barrier and is mediated by direct physical interactions among the macromolecules that form the condensate. We shall refer to this figure often as a guide in this chapter and the next, highlighting in color the particular pathway being discussed.
图 12-10 真核细胞内蛋白质运输的简化“路线图”。蛋白质可以通过蛋白质转位（蓝色）、门控运输（红色）、囊泡运输（绿色）或吞噬（灰色）从一个区室移动到另一个区室。指导特定蛋白质在系统中移动的排序信号，以及最终确定其在细胞中位置的信号，包含在每个蛋白质的氨基酸序列中。旅程始于在细胞质中的核糖体上合成蛋白质，对于许多蛋白质来说，当蛋白质到达其最终目的地时，旅程结束。其他蛋白质在细胞核和细胞质之间往返穿梭。在每个中间站（框）中，都会决定该蛋白质是保留在该区室内还是进一步运输。排序信号可以指示保留在区室内或从区室中退出。一种称为“吞噬”的特殊运输过程用于在自噬中将蛋白质从细胞质转移到溶酶体，或在有丝分裂后核膜重组期间将染色体包裹在细胞核内。大分子进出凝聚体的运动在此未显示。 这个过程不涉及穿越膜屏障，而是通过形成冷凝体的巨分子之间的直接物理相互作用来介导的。我们将在本章和下一章中经常引用这个图作为指南，并用颜色突出讨论的特定路径。

(Figure 12-11). The transfer of soluble and membrane-embedded proteins from the ER to the Golgi apparatus, for example, occurs in this way. The proteins transported by vesicular transport never cross a membrane during the process, and therefore retain their topological relationships within the cell.
（图 12-11）。可溶性和膜嵌入蛋白从内质网转移到高尔基体的过程就是以这种方式进行的。通过囊泡运输转运的蛋白在此过程中从未穿越膜，因此在细胞内保持其拓扑关系。
4. In engulfment, such as autophagy (discussed in Chapter 13), doublemembrane sheets wrap around portions of the cytoplasm often including fragments of organelles or even entire organelles (Figure 12-12). This membrane structure then seals by membrane fusion to enclose a separate compartment, the autophagosome. The re-formation of the nuclear envelope after mitosis (discussed later in this chapter) follows a conceptually similar process. ER tubes and sheets wrap around decondensing chromosomes and then fuse laterally with one another to form a sealed double-membrane envelope only traversed by the nuclear pores.
在吞噬过程中，例如自噬（在第 13 章中讨论），双膜片包裹细胞质的部分，通常包括细胞器的碎片或甚至整个细胞器（图 12-12）。该膜结构随后通过膜融合密封，形成一个独立的腔室，即自噬体。细胞分裂后核膜的重新形成（在本章后面讨论）遵循一个概念上类似的过程。内质网管和片包裹解旋的染色体，然后侧向融合形成一个仅由核孔穿越的密封双膜包膜。
In addition to these mechanisms for protein movement into and between membrane-enclosed compartments, a simpler mechanism based on direct physical binding is used by macromolecules to enter biomolecular condensates. In this mechanism, the macromolecule specifically binds to another protein or RNA that is already part of the condensate to which it is specifically recruited. Once recruited, the macromolecule remains within the condensate because of persistent and repeated interactions with its partner. The interaction between a macromolecule and its binding partner in the condensate is analogous to the interaction between a sorting signal and its cognate sorting receptor; in both cases, the interaction specifies the macromolecule’s destination.
除了这些将蛋白质转运到膜封闭的细胞器之间的机制外，大分子还使用一种基于直接物理结合的更简单机制进入生物分子凝聚体。在这一机制中，大分子特异性地结合到已经是其特定招募的凝聚体一部分的另一种蛋白质或 RNA 上。一旦被招募，大分子由于与其伙伴的持续和反复的相互作用而留在凝聚体内。大分子与其在凝聚体中的结合伙伴之间的相互作用类似于排序信号与其相应排序受体之间的相互作用；在这两种情况下，互动都指定了大分子的目的地。

Sorting Signals and Sorting Receptors Direct Proteins to the Correct Cell Address
排序信号和排序受体将蛋白质直接导向正确的细胞地址

Figure 12-11 Vesicle budding and fusion during vesicular transport. Transport vesicles bud from one compartment (donor) and fuse with another topologically equivalent (target) compartment. In the process, a subset of soluble components (red dots) are transferred from lumen to lumen. Note that membrane is also transferred and that the original orientation of both proteins and lipids in the donor compartment membrane is preserved in the target compartment membrane. Thus, membrane proteins retain their asymmetrical orientation, with the same domains always facing the cytosol.
图 12-11 泡囊运输过程中的泡囊出芽和融合。运输泡囊从一个腔室（供体）出芽，并与另一个拓扑等效的（靶）腔室融合。在此过程中，一部分可溶性成分（红点）从腔室转移到腔室。请注意，膜也被转移，并且供体腔室膜中蛋白质和脂质的原始取向在靶腔室膜中得以保留。因此，膜蛋白保持其不对称取向，相同的结构域始终面向细胞质。

import into nucleus
- Pro - Pro - Lys-Lys-Lys-Arg-Lys- Val -
export from nucleus
- Met - Glu - Glu - Leu - Ser - Gln - Ala - Leu - Ala - Ser - Ser - Phe -
import into mitochondria
    N - Met - Leu - Ser - Leu - Arg - Gln - Ser - Ile - Arg-Phe - Phe - Lys - Pro - Ala - Thr - Arg - Thr -
Leu - Cys - Ser - Ser - Arg- Tyr - Leu - Leu -
import into plastids
    N - Met - Val - Ala - Met - Ala - Met - Ala - Ser - Leu - Gln - Ser - Ser - Met - Ser - Ser - Leu - Ser -
    Leu - Ser - Ser - Asn - Ser - Phe - Leu - Gly - Gln - Pro - Leu - Ser - Pro - Ile - Thr - Leu - Ser - Pro -
    Phe - Leu - Gln - Gly -
import into peroxisomes
    -Ser-Lys-Leu-C
import into ER
    N - Met - Met - Ser - Phe - Val - Ser - Leu - Leu - Leu - Val - Gly - Ile - Leu - Phe - Trp - Ala - Thr -
    Glu - Ala - Glu - Gln - Leu - Thr - Lys - Cys - Glu - Val - Phe - Gln -

Construction of Most Organelles Requires Information in the Organelle Itself
大多数细胞器的构建需要细胞器自身的信息

Summary 摘要

During cell division, organelles such as the ER and mitochondria are distributed to each daughter cell. These organelles contain information that is required for their construction, and so they cannot be made de novo. Biomolecular condensates can be constructed de novo because they self-assemble from components that are encoded genetically.
在细胞分裂过程中，内质网和线粒体等细胞器被分配到每个子细胞中。这些细胞器包含其构建所需的信息，因此无法通过新合成的方式制造。生物分子凝聚体可以通过新合成的方式构建，因为它们是由基因编码的成分自组装而成的。

THE ENDOPLASMIC RETICULUM
内质网

The ER Is Structurally and Functionally Diverse
内质网在结构和功能上具有多样性

Figure 12-14 Fluorescence micrographs of the endoplasmic reticulum. (A) An animal cell in tissue culture that was genetically engineered to express an ER membrane protein fused to a fluorescent protein. The ER extends as a network of tubules and sheets throughout the entire cytosol, so that all regions of the cytosol are close to some portion of the ER membrane. The outer nuclear membrane, which is continuous with the $ER$$ER$ERE R, is also stained. (B) Part of an ER network in a living plant cell that was genetically engineered to express a fluorescent protein in the ER. (A, courtesy of Patrick Chitwood and Gia Voeltz. B, courtesy of Petra Boevink and Chris Hawes.)
图 12-14 内质网的荧光显微照片。(A) 一种在组织培养中经过基因工程改造的动物细胞，表达与荧光蛋白融合的内质网膜蛋白。内质网在整个细胞质中延伸为管状和片状的网络，使得细胞质的所有区域都靠近内质网膜的某个部分。外核膜与 $ER$$ER$ERE R 相连，也被染色。(B) 一种在活植物细胞中经过基因工程改造的内质网网络部分，该细胞在内质网中表达荧光蛋白。(A，感谢 Patrick Chitwood 和 Gia Voeltz 提供。B，感谢 Petra Boevink 和 Chris Hawes 提供。)

Another crucially important function of the ER in most eukaryotic cells is to sequester ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} from the cytosol. The release of ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} into the cytosol from the
大多数真核细胞中内质网的另一个至关重要的功能是将 ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} 从细胞质中隔离。 ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} 从细胞质中释放的过程

To study the functions and biochemistry of the ER, it is necessary to isolate it. This may seem to be a hopeless task because the ER is intricately interleaved with other components of the cytoplasm. Fortunately, when tissues or cells are disrupted by homogenization, the ER breaks into fragments, which reseal to form small ( $\sim 100-200\mathrm{nm}$$\sim 100-200\mathrm{nm}$∼100-200nm\sim 100-200 \mathrm{~nm} in diameter) closed vesicles called microsomes (Figure 12-17). To the biochemist, microsomes represent small authentic versions of the ER, still capable of protein translocation, protein glycosylation (discussed later), ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} uptake and release, and lipid synthesis. Rough microsomes, derived from rough ER, contain ribosomes on their outside surface and enclose a small part of the ER lumen. Smooth microsomes, which lack ribosomes, are derived from vesiculated fragments of the smooth ER, plasma membrane, Golgi apparatus, endosomes, and mitochondria. The ribosomes attached to rough microsomes make them denser than smooth microsomes. As a result, scientists use equilibrium density centrifugation to separate the rough and smooth microsomes (Figure 12-17). Smooth microsomes derived from different organelles can
为了研究内质网的功能和生物化学，必须对其进行分离。这似乎是一项绝望的任务，因为内质网与细胞质的其他成分错综复杂地交织在一起。幸运的是，当组织或细胞通过匀浆破坏时，内质网会断裂成碎片，这些碎片会重新封闭形成直径约为 $\sim 100-200\mathrm{nm}$$\sim 100-200\mathrm{nm}$∼100-200nm\sim 100-200 \mathrm{~nm} 的小闭合囊泡，称为微粒体（图 12-17）。对于生物化学家来说，微粒体代表了内质网的小型真实版本，仍然能够进行蛋白质转运、蛋白质糖基化（后文讨论）、 ${\mathrm{Ca}}^{2+}$${\mathrm{Ca}}^{2+}$Ca^(2+)\mathrm{Ca}^{2+} 的摄取和释放以及脂质合成。粗微粒体来源于粗内质网，外表面含有核糖体，并包裹着内质网腔的一小部分。光滑微粒体则缺乏核糖体，来源于光滑内质网、质膜、高尔基体、内体和线粒体的囊泡化碎片。附着在粗微粒体上的核糖体使其比光滑微粒体更密集。因此，科学家们使用平衡密度离心法来分离粗微粒体和光滑微粒体（图 12-17）。来源于不同细胞器的光滑微粒体可以

Signal Sequences Were First Discovered in Proteins Imported into the Rough ER
信号序列最早是在导入粗糙内质网的蛋白质中发现的

Signal sequences (and the signal sequence strategy of protein sorting) were discovered in secreted water-soluble proteins that are first translocated across the ER membrane. In the key experiment, the mRNA encoding a secreted protein was added to cytosol extracted from cells. In this cell-free reaction, ribosomes in the cytosol translated the mRNA into a protein that was slightly larger than the normal secreted protein (Figure 12-18). When the reaction was repeated in the presence of microsomes derived from the rough ER, a protein of the correct size was produced and located inside the microsomes (Figure 12-18). By contrast, mRNA encoding a cytosolic protein produced the correctly sized product regardless of the presence or absence of rough microsomes. The signal hypothesis was formulated to explain these observations. According to this model, the mRNA for the secretory protein codes for a protein that is bigger than the protein that is eventually secreted. It was proposed that the extra polypeptide is a signal sequence that directs the secreted protein to the ER membrane. After the signal sequence has served its function, it is cleaved off by a signal peptidase in the ER membrane before the polypeptide chain has been completed.
信号序列（以及蛋白质分选的信号序列策略）是在分泌的水溶性蛋白质中发现的，这些蛋白质首先被转运穿过内质网膜。在关键实验中，编码分泌蛋白的 mRNA 被添加到从细胞提取的细胞质中。在这个无细胞反应中，细胞质中的核糖体将 mRNA 翻译成一种比正常分泌蛋白稍大的蛋白质（图 12-18）。当在来源于粗糙内质网的微粒体存在下重复该反应时，产生了正确大小的蛋白质，并位于微粒体内部（图 12-18）。相比之下，编码细胞质蛋白的 mRNA 无论在粗糙微粒体的存在与否下都产生了正确大小的产物。信号假说被提出以解释这些观察结果。根据该模型，分泌蛋白的 mRNA 编码的蛋白质比最终分泌的蛋白质要大。有人提出，额外的多肽是一个信号序列，指引分泌蛋白到达内质网膜。 在信号序列完成其功能后，它在内质网膜上被信号肽酶切除，此时多肽链尚未完成。

A Signal-Recognition Particle (SRP) Directs the ER Signal Sequence to a Specific Receptor at the ER
信号识别颗粒（SRP）将内质网信号序列导向内质网特定受体

The ER signal sequence is guided to the ER membrane by at least two components: a signal-recognition particle (SRP), which binds to the signal sequence, and an SRP receptor in the ER membrane. SRP is a large complex; in animal cells, it consists of six different polypeptide chains bound to a single RNA molecule (Figure 12-19A). While SRP and SRP receptor have fewer subunits in bacteria, homologs of both components are present in all living organisms.
内质网信号序列通过至少两个组分引导到内质网膜：一个信号识别颗粒（SRP），它与信号序列结合，以及内质网膜中的 SRP 受体。SRP 是一个大型复合体；在动物细胞中，它由六条不同的多肽链与一个单一的 RNA 分子结合（图 12-19A）。虽然 SRP 和 SRP 受体在细菌中具有较少的亚基，但这两个组分的同源物在所有生物中都存在。

Because many ribosomes can engage with a single mRNA molecule, a polyribosome is usually formed. If the mRNA encodes a protein with an ER signal sequence, the polyribosome becomes attached to the ER membrane, directed there by the signal sequences on multiple growing polypeptide chains. The individual ribosomes associated with such an mRNA molecule can return to the cytosol when they finish translation and intermix with the pool of free ribosomes. The mRNA itself, however, remains attached to the ER membrane by a
由于许多核糖体可以与单个 mRNA 分子结合，因此通常会形成多核糖体。如果 mRNA 编码一个带有内质网信号序列的蛋白质，则多核糖体会附着在内质网膜上，受到多个正在生长的多肽链上的信号序列的引导。与这种 mRNA 分子相关的单个核糖体在完成翻译后可以返回细胞质，并与游离核糖体的池混合。然而，mRNA 本身仍然附着在内质网膜上。

Figure 12-20 How ER signal sequences and SRP direct ribosomes to the ER membrane. The SRP and its receptor act in concert. The SRP binds to both the exposed ER signal sequence and the ribosome, thereby causing translation to slow. The SRP receptor in the ER membrane, which in animal cells is composed of two different polypeptide chains, binds the SRP-ribosome complex and directs it to the translocator. The SRP (in complex with SRP receptor) then moves away from its binding site on the ribosome, which is then occupied by the translocator in the ER membrane. SRP then releases the signal sequence, which inserts into the translocator to initiate polypeptide chain transfer across the lipid bilayer. The SRP and SRP receptor dissociate from each other and are recycled for the next round of protein targeting. Although not shown in the figure, one of the SRP proteins and both chains of the SRP receptor contain GTP-binding domains. Conformational changes that occur during cycles of GTP binding and hydrolysis (discussed in Chapter 15) ensure that SRP preferentially binds a signal sequence in the cytosol and releases it only after SRP successfully engages the SRP receptor at the ER membrane. The energy of GTP hydrolysis is therefore used to impart directionality to the cycle of SRP-mediated protein targeting.
图 12-20 ER 信号序列和 SRP 如何引导核糖体到达 ER 膜。SRP 及其受体协同作用。SRP 同时与暴露的 ER 信号序列和核糖体结合，从而导致翻译减缓。ER 膜中的 SRP 受体，在动物细胞中由两条不同的多肽链组成，结合 SRP-核糖体复合物并将其引导至转运蛋白。SRP（与 SRP 受体复合）随后从核糖体上的结合位点移开，该位点随后被 ER 膜中的转运蛋白占据。SRP 随后释放信号序列，信号序列插入转运蛋白中以启动多肽链跨越脂质双层的转移。SRP 和 SRP 受体相互解离，并被回收以进行下一轮的蛋白质靶向。尽管图中未显示，SRP 蛋白中的一个和 SRP 受体的两个链都含有 GTP 结合域。在 GTP 结合和水解周期中发生的构象变化（在第 15 章中讨论）确保 SRP 优先在细胞质中结合信号序列，并仅在 SRP 成功与 ER 膜上的 SRP 受体结合后释放该信号序列。 GTP 水解的能量因此被用来为 SRP 介导的蛋白质靶向循环赋予方向性。

The Polypeptide Chain Passes Through a Signal Sequence-gated Aqueous Channel in the Translocator
多肽链通过转运蛋白中的信号序列门控水相通道

The Sec61 translocator only opens for proteins containing a signal sequence. The ability of the Sec61 translocator to recognize signal sequences provides a proofreading step to ensure that only proteins truly intended for the ER are
Sec61 转运蛋白仅对含有信号序列的蛋白质开放。Sec61 转运蛋白识别信号序列的能力提供了一个校对步骤，以确保只有真正打算进入内质网的蛋白质被转运。

When translation terminates, the C-terminus of the polypeptide is released from the ribosome and slips through the Sec61 translocator, whose plug returns to close the channel. Thus, the entire process of ER import, from signal sequence recognition by SRP to translocation through the Sec61 translocator, occurs co-translationally before the polypeptide has a chance to fold. This pathway provides one solution
当翻译终止时，多肽的 C 末端从核糖体释放，并通过 Sec61 转运蛋白滑入，塞子返回以关闭通道。因此，从信号序列被 SRP 识别到通过 Sec61 转运蛋白转位的整个内质网进口过程，都是在多肽有机会折叠之前共同翻译进行的。这条途径提供了一种解决方案。

Translocation Across the ER Membrane Does Not Always Require Ongoing Polypeptide Chain Elongation
跨内质网膜的转位并不总是需要持续的多肽链延伸

Some proteins are completely synthesized in the cytosol as precursors before they are imported into the ER, demonstrating that translocation does not always require ongoing translation (Figure 12-24). This is termed post-translational translocation. Post-translational protein translocation is more common across the yeast ER membrane and the evolutionarily related bacterial plasma membrane. In both cases, the Sec61 translocator (called SecY in bacteria) is used as the
一些蛋白质在细胞质中作为前体完全合成后再被导入内质网，这表明转位并不总是需要持续的翻译（图 12-24）。这被称为翻译后转位。翻译后蛋白质转位在酵母内质网膜和进化相关的细菌质膜中更为常见。在这两种情况下，使用 Sec61 转位蛋白（在细菌中称为 SecY）。

Figure 12-24 Co-translational and post-translational protein translocation. Ribosomes bind to the ER membrane during co-translational translocation. By contrast, cytosolic ribosomes complete the synthesis of a protein and release it prior to post-translational translocation. The released protein is kept unfolded in the cytosol by chaperones that dissociate before the protein is translocated across the membrane. In both cases, the protein is directed to the ER by an ER signal sequence (red and orange). See Movie 12.3.
图 12-24 共同翻译和翻译后蛋白质转位。核糖体在共同翻译转位过程中与内质网膜结合。相比之下，细胞质中的核糖体在翻译后转位之前完成蛋白质的合成并释放它。释放的蛋白质在细胞质中由伴侣蛋白保持未折叠状态，这些伴侣蛋白在蛋白质跨膜转位之前解离。在这两种情况下，蛋白质都通过内质网信号序列（红色和橙色）被导向内质网。请参见电影 12.3。

Transmembrane Proteins Contain Hydrophobic Segments That Are Recognized Like Signal Sequences
跨膜蛋白含有被识别为信号序列的疏水片段

Figure 12-26 A transmembrane segment directs membrane protein insertion into the ER membrane. Many single-pass membrane proteins use their transmembrane segment to direct insertion into the ER membrane (Movie 12.3). The transmembrane segment is recognized by SRP (not shown) and delivered via the SRP receptor (not shown) to the Sec61 translocator at the ER membrane. The transmembrane segment then inserts into the lateral gate of the Sec61 translocator in one of two orientations. (A) Some transmembrane segments insert into the lateral gate such that the N-terminal domain is retained on the cytosolic side of Sec61. This orientation is favored for proteins whose N -terminal domains are very long or folded, and for transmembrane segments whose flanking amino acids have a net positive charge on the N -terminal side. (B) Some transmembrane segments insert into the lateral gate such that the C-terminal flanking region is retained on the cytosolic side of Sec61. In this case, the N-terminal flanking region is thought to translocate across the membrane through the Sec61 channel. This orientation is favored for transmembrane segments whose flanking amino acids have a net positive charge on the C-terminal side.
图 12-26 跨膜片段引导膜蛋白插入内质网膜。许多单跨膜蛋白利用其跨膜片段引导插入内质网膜（电影 12.3）。跨膜片段被信号识别颗粒（SRP，未显示）识别，并通过 SRP 受体（未显示）送至内质网膜上的 Sec61 转运蛋白。然后，跨膜片段以两种取向之一插入 Sec61 转运蛋白的侧门。（A）一些跨膜片段插入侧门，使得 N 端结构保留在 Sec61 的细胞质侧。这种取向适用于 N 端结构非常长或折叠的蛋白质，以及 N 端侧的邻近氨基酸具有净正电荷的跨膜片段。（B）一些跨膜片段插入侧门，使得 C 端邻近区域保留在 Sec61 的细胞质侧。在这种情况下，N 端邻近区域被认为是通过 Sec61 通道跨膜转运的。 这种取向适用于其侧翼氨基酸在 C 端具有净正电荷的跨膜片段。

mature single-pass transmembrane
成熟的单通道跨膜
protein in ER membrane
内质网膜中的蛋白质

utilize both a cleaved ER signal sequence and a transmembrane segment. Targeting to the ER membrane, initiation of translocation through Sec61, and cleavage of the signal sequence all occur exactly as for a secretory protein (see Figure 12-20). However, when the transmembrane segment enters the Sec61 translocator, translocation stops and the transmembrane segment moves through the lateral gate into the lipid bilayer. The remainder of the protein continues to be synthesized on the cytosolic side of the membrane until translation terminates.
利用切割的内质网信号序列和跨膜片段。靶向内质网膜、通过 Sec61 的转位启动以及信号序列的切割都与分泌蛋白完全相同（见图 12-20）。然而，当跨膜片段进入 Sec61 转位器时，转位停止，跨膜片段通过侧门移动到脂质双层中。蛋白质的其余部分继续在膜的细胞质侧合成，直到翻译终止。

Hydrophobic Segments of Multipass Transmembrane Proteins Are Interpreted Contextually to Determine Their Orientation
多跨膜蛋白的疏水片段通过上下文进行解读以确定其取向

Because of the tight coupling between the ribosome and Sec61 translocator, each hydrophobic segment emerges very close to the lateral gate that provides access to the lipid bilayer. In the simplest cases, the newly emerged hydrophobic segment engages the lateral gate in an orientation opposite to the most recently inserted transmembrane segment and inserts into the lipid bilayer (Figure 12-28). Some transmembrane segments of multipass proteins are only partially hydrophobic and would not be stable in the lipid bilayer on their own. These can nevertheless insert into the membrane if they are able to interact with one of the preceding transmembrane segments that is near the lateral gate of Sec61. This cooperation makes it possible to produce multipass transmembrane proteins that contain hydrophilic parts within the lipid bilayer, which is crucial
由于核糖体与 Sec61 转运蛋白之间的紧密耦合，每个疏水段都会非常接近提供进入脂质双层的侧门。在最简单的情况下，新出现的疏水段以与最近插入的跨膜段相反的方向与侧门结合，并插入脂质双层（图 12-28）。一些多跨膜蛋白的跨膜段仅部分疏水，单独在脂质双层中不稳定。然而，如果它们能够与靠近 Sec61 侧门的前一个跨膜段相互作用，它们仍然可以插入膜中。这种协作使得能够产生在脂质双层中含有亲水部分的多跨膜蛋白，这一点至关重要。

Some Proteins Are Integrated into the ER Membrane by a Post-translational Mechanism
某些蛋白质通过后翻译机制整合到内质网膜中

Many important cytosol-facing membrane proteins are anchored in the membrane by a single transmembrane $\alpha$$\alpha$alpha\alpha helix very close to the C-terminus. These tail-anchored proteins include a large number of SNARE protein subunits that guide vesicular traffic (discussed in Chapter 13). When a tail-anchored protein is translated, the ribosome reaches the termination codon while the polypeptide sequence destined to become a transmembrane $\alpha$$\alpha$alpha\alpha helix is still inside the ribosome exit tunnel. Recognition by SRP is therefore not possible, and the protein is released from the ribosome into the cytosol. The hydrophobic segment is recognized by a specialized chaperone complex that transfers it to a targeting factor called Get3 (Figure 12-29). Although unrelated to SRP, Get3 also contains a hydrophobic pocket lined by many methionine side chains to help it recognize diverse hydrophobic segments independent of their exact sequence. Two proteins at the ER membrane called Getl and Get 2 serve not only as the receptor for Get3 but also as the translocator that inserts the hydrophobic segment of the tail-anchored protein into the lipid bilayer. This post-translational targeting mechanism is therefore conceptually similar to SRP-dependent protein targeting (see Figure 12-20). Some tail-anchored proteins are targeted to mitochondria or peroxisomes instead of the ER, but the mechanism of their targeting is not known.
许多重要的面向细胞质的膜蛋白通过一个非常接近 C 末端的单个跨膜 $\alpha$$\alpha$alpha\alpha 螺旋锚定在膜中。这些尾锚定蛋白包括大量的 SNARE 蛋白亚基，它们引导囊泡运输（在第 13 章中讨论）。当尾锚定蛋白被翻译时，核糖体在多肽序列仍在核糖体出口通道内时到达终止密码子。因此，SRP 的识别是不可能的，蛋白质从核糖体释放到细胞质中。疏水段被一个专门的伴侣复合物识别，该复合物将其转移到一个称为 Get3 的靶向因子（图 12-29）。尽管与 SRP 无关，Get3 也包含一个由许多蛋氨酸侧链衬里的疏水口袋，以帮助其独立于确切序列识别多样的疏水段。位于内质网膜上的两个蛋白质 Get1 和 Get2 不仅作为 Get3 的受体，还作为转位体，将尾锚定蛋白的疏水段插入脂质双层中。 因此，这种翻译后靶向机制在概念上类似于 SRP 依赖的蛋白质靶向（见图 12-20）。一些尾锚定蛋白被靶向到线粒体或过氧化物酶体，而不是内质网，但其靶向机制尚不清楚。

Some Membrane Proteins Acquire a Covalently Attached Glycosylphosphatidylinositol (GPI) Anchor
某些膜蛋白获得共价附着的糖苷磷脂酰肌醇（GPI）锚

Translocated Polypeptide Chains Fold and Assemble in the Lumen of the Rough ER
转位多肽链在粗糙内质网的腔内折叠和组装

Proteins enter the ER lumen as unfolded polypeptides. They must therefore fold and assemble into their correct three-dimensional structures just as newly made proteins in the cytosol must fold (discussed in Chapter 3). To meet this demand, the lumen of the ER contains a high concentration of resident chaperones and other protein-folding catalysts. These ER resident proteins contain an ER retention signal of four amino acids at their C-terminus that is responsible for retaining the protein in the ER (see Figure 12-13; discussed in Chapter 13, p. 768).
蛋白质以未折叠的多肽形式进入内质网腔。因此，它们必须折叠并组装成正确的三维结构，就像细胞质中新合成的蛋白质必须折叠一样（在第 3 章中讨论）。为了满足这一需求，内质网腔中含有高浓度的驻留伴侣蛋白和其他蛋白质折叠催化剂。这些内质网驻留蛋白在其 C 末端含有一个由四个氨基酸组成的内质网保留信号，负责将蛋白质保留在内质网中（见图 12-13；在第 13 章，第 768 页讨论）。

The ER resident protein protein disulfide isomerase (PDI) catalyzes the oxidation of free sulfhydryl (SH) groups on cysteines to form disulfide (S-S) bonds (Figure 12-31). Almost all cysteines in protein domains exposed to either the
内质网驻留蛋白质二硫键异构酶（PDI）催化游离巯基（SH）在半胱氨酸上的氧化，形成二硫键（S-S）（图 12-31）。几乎所有暴露于蛋白质结构域的半胱氨酸都参与了这一过程。

Most Proteins Synthesized in the Rough ER Are Glycosylated by the Addition of a Common N-Linked Oligosaccharide
大多数在粗糙内质网合成的蛋白质通过添加一种共同的 N-连接寡糖进行糖基化

During the most common form of protein glycosylation in the ER, a preformed precursor oligosaccharide (containing 14 sugars composed of 2 N acetylglucosamines, 9 mannoses, and 3 glucoses) is transferred as a complete unit to proteins. Because this oligosaccharide is transferred to the side-chain ${\mathrm{NH}}_2$${\mathrm{NH}}_2$NH_(2)\mathrm{NH}_{2} group of an asparagine in the protein, it is said to be N -linked, or asparagine-linked (Figure 12-32A). A special lipid molecule called dolichol (see Panel 2-5, pp. 102-103) anchors the precursor oligosaccharide in the ER membrane. The precursor oligosaccharide is transferred to the target asparagine in a single enzymatic step by an oligosaccharyl transferase. This membrane-bound enzyme associates with the Sec61 translocator and has its active site exposed on the lumenal side
在内质网中最常见的蛋白质糖基化形式中，一个预先形成的前体寡糖（包含 14 个糖，由 2 个 N-乙酰氨基葡萄糖、9 个甘露糖和 3 个葡萄糖组成）作为一个完整的单元转移到蛋白质上。由于这个寡糖被转移到蛋白质中天冬氨酸的侧链 ${\mathrm{NH}}_2$${\mathrm{NH}}_2$NH_(2)\mathrm{NH}_{2} 基团上，因此称为 N-连接或天冬氨酸连接（图 12-32A）。一种叫做多烯醇的特殊脂质分子（见面板 2-5，第 102-103 页）将前体寡糖锚定在内质网膜上。前体寡糖通过一种寡糖转移酶在单一的酶促步骤中转移到目标天冬氨酸上。这个膜结合的酶与 Sec61 转运蛋白结合，并且其活性位点暴露在腔内侧。

Figure 12-32 $N$$N$NN-linked protein glycosylation in the rough ER. (A) Almost as soon as a polypeptide chain enters the ER lumen, it is glycosylated on target asparagine amino acids. The precursor oligosaccharide (shown in color) is attached only to asparagine side chains in the sequences Asn-X-Ser and Asn-X-Thr (where $X$$X$XX is any amino acid except proline). These sequences occur much less frequently in glycoproteins than in nonglycosylated cytosolic proteins. Evidently there has been selective pressure against these sequences during protein evolution, presumably because glycosylation at inappropriate sites would interfere with protein folding. The five sugars in the gray box form the core region of this oligosaccharide. For many glycoproteins, only the core sugars survive the extensive oligosaccharide trimming that takes place in the Golgi apparatus (Movie 13.4). (B) The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane oligosaccharyl transferase enzyme complex. One copy of this enzyme is associated with each protein translocator in the ER membrane. Oligosaccharyl transferase contains 13 transmembrane $\alpha$$\alpha$alpha\alpha helices and a large ER lumenal domain that contains binding sites for the nascent protein and dolichol-oligosaccharide. The asparagine binds a tunnel that penetrates the enzyme interior. There, the amino group of the asparagine is twisted out of the plane that stabilizes the otherwise poorly reactive amide bond, activating it for reaction with the dolichol-oligosaccharide.
图 12-32 $N$$N$NN - 粗糙内质网中的蛋白质糖基化。(A) 一旦多肽链进入内质网腔，它几乎立即在目标天冬氨酸氨基酸上进行糖基化。前体寡糖（以颜色显示）仅附着在序列 Asn-X-Ser 和 Asn-X-Thr 中的天冬氨酸侧链上（其中 $X$$X$XX 是除脯氨酸以外的任何氨基酸）。这些序列在糖蛋白中出现的频率远低于在非糖基化的细胞质蛋白中。显然，在蛋白质进化过程中，这些序列受到选择压力，可能是因为在不适当的位置进行糖基化会干扰蛋白质折叠。灰色框中的五种糖形成了该寡糖的核心区域。对于许多糖蛋白来说，只有核心糖在高尔基体中进行的广泛寡糖修剪中存活下来（电影 13.4）。(B) 前体寡糖作为一个完整的单元从多烯醇脂质锚转移到天冬氨酸上，这一反应由跨膜寡糖基转移酶酶复合物催化。每个蛋白质转运体与内质网膜中的一个酶副本相关联。 寡糖基转移酶包含 13 个跨膜 $\alpha$$\alpha$alpha\alpha 螺旋和一个大的内质网腔域，该域包含新生蛋白和多烯醇-寡糖的结合位点。天冬酰胺结合一个穿透酶内部的通道。在那里，天冬酰胺的氨基被扭转出稳定原本反应性较差的酰胺键的平面，从而激活其与多烯醇-寡糖反应。

of the ER membrane. This allows the oligosaccharyl transferase to modify newly made proteins immediately after the target asparagine enters the ER lumen during protein translocation (Figure 12-32B).
内质网膜的。这使得寡糖基转移酶能够在目标天冬氨酸进入内质网腔体后立即修饰新合成的蛋白质，发生在蛋白质转运过程中（图 12-32B）。

Oligosaccharides Are Used as Tags to Mark the State of Protein Folding
寡糖被用作标记蛋白质折叠状态的标签

Figure 12-33 Synthesis of the lipidlinked precursor oligosaccharide in the rough ER membrane. The oligosaccharide is assembled sugar by sugar onto the carrier lipid dolichol (a polyisoprenoid; see Panel 2-5, pp. 102-103). Dolichol is long and very hydrophobic: its 22 fivecarbon units can span the thickness of a lipid bilayer more than three times, so that the attached oligosaccharide is firmly anchored in the membrane. The first sugar is linked to dolichol by a pyrophosphate bridge. This high-energy bond activates the oligosaccharide for its eventual transfer from the lipid to an asparagine side chain of a growing polypeptide on the lumenal side of the rough ER. As indicated, the synthesis of the oligosaccharide starts on the cytosolic side of the ER membrane and continues on the lumenal face after the $(\mathrm{Man}{)}_5(\mathrm{GlcNAc}{)}_2$$(\mathrm{Man}{)}_5(\mathrm{GlcNAc}{)}_2$(Man)_(5)(GlcNAc)_(2)(\mathrm{Man})_{5}(\mathrm{GlcNAc})_{2} lipid intermediate is flipped across the bilayer by a transporter (which is not shown). All the subsequent glycosyl transfer reactions on the lumenal side of the ER involve transfers from dolichol-P-glucose and dolichol-Pmannose; these activated, lipid-linked monosaccharides are synthesized from dolichol phosphate and UDP-glucose or GDP-mannose (as appropriate) on the cytosolic side of the ER and are then flipped across the ER membrane. GIcNAc $=N$$=N$=N=N-acetylglucosamine; Man $=$$=$== mannose; Glc = glucose.
图 12-33 在粗糙内质网膜中合成脂质连接前体寡糖。寡糖是一个个糖分子组装到载体脂质多烯醇（聚异戊二烯；见面板 2-5，第 102-103 页）上。多烯醇长且非常疏水：其 22 个五碳单位可以跨越脂质双层的厚度超过三倍，因此附着的寡糖牢牢固定在膜中。第一个糖通过焦磷酸桥连接到多烯醇上。这个高能键激活了寡糖，以便最终将其从脂质转移到粗糙内质网腔侧生长多肽的天冬氨酸侧链上。如所示，寡糖的合成始于内质网膜的细胞质侧，并在脂质中间体通过转运蛋白（未显示）翻转到双层的腔侧后继续进行。 所有后续的糖基转移反应发生在内质网的腔侧，涉及从多烯醇-P-葡萄糖和多烯醇-P-甘露糖的转移；这些活化的脂质连接单糖是由多烯醇磷酸盐和 UDP-葡萄糖或 GDP-甘露糖（视情况而定）在内质网的细胞质侧合成的，然后被翻转穿过内质网膜。GIcNAc $=N$$=N$=N=N -乙酰氨基葡萄糖；Man $=$$=$== 甘露糖；Glc = 葡萄糖。

and retain them in the ER. Like other chaperones, they prevent incompletely folded proteins from irreversibly aggregating. Both calnexin and calreticulin also promote the association of incompletely folded proteins with another ER chaperone, which binds to cysteines that have not yet formed disulfide bonds.
并将它们保留在内质网中。像其他伴侣蛋白一样，它们防止未完全折叠的蛋白质不可逆地聚集。卡尔内克辛和卡雷图林还促进未完全折叠的蛋白质与另一种内质网伴侣蛋白的结合，该伴侣蛋白与尚未形成二硫键的半胱氨酸结合。

Improperly Folded Proteins Are Exported from the ER and Degraded in the Cytosol
不正确折叠的蛋白质从内质网被转运并在细胞质中降解

Figure 12-34 The role of $N$$N$NN-linked glycosylation in ER protein folding. The ER membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on $N$$N$NN-linked oligosaccharides, trapping the protein in the ER. Removal of the terminal glucose by a glucosidase releases the protein from calnexin. A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the $N$$N$NN-linked oligosaccharide, renewing the protein’s affinity for calnexin and retaining it in the ER. The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble ER resident protein. Another ER chaperone, ERp57 (not shown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER. ERp57 recognizes free sulfhydryl groups, which are a sign of incomplete disulfide bond formation. The longer a protein spends in this cycle without folding correctly, the more likely it is that ERresident mannosidase enzymes (not shown) remove the terminal mannoses from the $N$$N$NN-linked oligosaccharide. The trimmed oligosaccharide with reduced mannoses is recognized by other ER lectins that route the polypeptide for degradation. Thus, only proteins that fold promptly and exit the ER avoid trimming by mannosidases and escape degradation.
图 12-34 $N$$N$NN -连接糖基化在内质网蛋白折叠中的作用。内质网膜结合的伴侣蛋白 calnexin 与含有一个末端葡萄糖的 $N$$N$NN -连接寡糖的不完全折叠蛋白结合，将其困在内质网中。通过葡萄糖苷酶去除末端葡萄糖后，蛋白质从 calnexin 中释放出来。葡萄糖转移酶是决定蛋白质是否正确折叠的关键酶：如果蛋白质仍然是不完全折叠的，该酶会将 UDP-葡萄糖中的新葡萄糖转移到 $N$$N$NN -连接寡糖上，更新蛋白质与 calnexin 的亲和力并将其保留在内质网中。该循环重复，直到蛋白质完全折叠。calreticulin 的功能类似，只是它是一种可溶性内质网驻留蛋白。另一个内质网伴侣蛋白 ERp57（未显示）与 calnexin 和 calreticulin 协作，将不完全折叠的蛋白质保留在内质网中。ERp57 识别游离巯基，这表明二硫键形成不完全。 蛋白质在这个循环中未正确折叠的时间越长，ER 驻留的甘露糖苷酶（未显示）去除 $N$$N$NN -连接寡糖的末端甘露糖的可能性就越大。经过修剪的寡糖由于甘露糖减少而被其他 ER 凝集素识别，这些凝集素将多肽引导至降解。因此，只有及时折叠并离开内质网的蛋白质才能避免被甘露糖苷酶修剪并逃避降解。

Misfolded Proteins in the ER Activate an Unfolded Protein Response
内质网中的错误折叠蛋白激活未折叠蛋白反应

Figure 12-35 The export and degradation of misfolded ER proteins. Misfolded soluble proteins in the ER lumen are recognized and targeted to a translocator complex in the ER membrane. They first interact in the ER lumen with chaperones, disulfide isomerases, and lectins. The chaperones maintain the misfolded protein in an unfolded conformation and prevent their aggregation. The disulfide isomerases reduce disulfide bonds to fully unfold the protein. The lectins selectively recognize trimmed $N$$N$NN-linked oligosaccharides that are generated when a protein spends too long in the ER. The lectins have binding sites on a membrane-embedded protein translocator built around an E3 ubiquitin ligase. The unfolded protein is then exported into the cytosol through the translocator. The E3 ubiquitin ligase ubiquitylates the unfolded protein as it emerges on the cytosolic side of the translocator. The ubiquitin prevents backsliding of the protein into the ER and provides a molecular handle for an AAA-ATPase that completes the extraction reaction. The unfolded protein is then de-glycosylated and degraded in proteasomes. Misfolded membrane proteins follow a similar pathway but are thought to engage the translocator sideways within the lipid bilayer. Multiple translocator complexes containing different E3 ubiquitin ligases reside in the ER. They are thought to handle different subsets of proteins that are misfolded in different ways.
图 12-35 错误折叠内质网蛋白的出口与降解。内质网腔中的错误折叠可溶性蛋白被识别并靶向到内质网膜中的转运复合体。它们首先在内质网腔中与伴侣蛋白、二硫键异构酶和凝集素相互作用。伴侣蛋白保持错误折叠蛋白处于未折叠构象，并防止其聚集。二硫键异构酶还原二硫键以完全展开蛋白。凝集素选择性识别在蛋白质在内质网中停留过久时生成的修剪过的 $N$$N$NN -连接寡糖。凝集素在围绕 E3 泛素连接酶构建的膜嵌入蛋白转运体上具有结合位点。未折叠的蛋白随后通过转运体被输出到细胞质中。E3 泛素连接酶在未折叠蛋白从转运体的细胞质侧出现时对其进行泛素化。泛素防止蛋白质回流到内质网，并为完成提取反应的 AAA-ATP 酶提供了分子把手。未折叠的蛋白随后被去糖基化并在蛋白酶体中降解。 错误折叠的膜蛋白遵循类似的途径，但被认为是在脂质双层内侧向地与转运蛋白结合。含有不同 E3 泛素连接酶的多个转运复合物存在于内质网中。它们被认为处理以不同方式错误折叠的不同蛋白质子集。

protein response, which stimulates transcription of genes that collectively improve the protein-folding capacity of the ER. The stimulated genes code for ER chaperones, the machinery for protein retrotranslocation and degradation, factors for protein transport out of the ER, and factors for expansion of the ER. This multipronged response operates by coupling the detection of misfolded proteins in the ER lumen to the production of transcription regulatory proteins that enter the nucleus to tune the transcription of hundreds of genes.
蛋白质反应，刺激基因的转录，这些基因共同提高内质网的蛋白质折叠能力。被刺激的基因编码内质网伴侣蛋白、蛋白质逆转运和降解的机械装置、将蛋白质运输出内质网的因子，以及扩展内质网的因子。这种多管齐下的反应通过将内质网腔内错误折叠蛋白的检测与转录调节蛋白的产生相结合来运作，这些转录调节蛋白进入细胞核以调节数百个基因的转录。

Figure 12-36 The unfolded protein response. Three parallel intracellular signaling pathways sense misfolded proteins in the ER lumen and lead to the activation of transcription in the nucleus. Each pathway begins with an ER-resident sensor of misfolded proteins. When these sensors are activated, they initiate different downstream signaling pathways. Although the downstream mechanisms are very different from each other, all of them culminate with the production of an active transcription factor. The overlapping targets of the transcription factors produce gene products that improve the proteinprocessing capacity of the ER and increase the protein degradation capacity of the cell.
图 12-36 展开蛋白质反应。三个平行的细胞内信号通路感知内质网腔中的错误折叠蛋白，并导致核内转录的激活。每个通路都以内质网驻留的错误折叠蛋白传感器开始。当这些传感器被激活时，它们会启动不同的下游信号通路。尽管下游机制彼此非常不同，但它们都以产生活性转录因子为最终结果。转录因子的重叠靶标产生的基因产物提高了内质网的蛋白质处理能力，并增加了细胞的蛋白质降解能力。

activate the transcription of genes in the nucleus. When misfolded proteins accumulate in the ER, the ATF6 protein is transported to the Golgi apparatus. Resident proteases in the Golgi apparatus membrane cleave off the cytosolic domain of ATF6, which can now migrate to the nucleus and help activate the transcription of genes encoding proteins involved in the unfolded protein response. This mechanism of activation of a latent membrane-embedded transcription factor is similar to how the transcription regulator that controls cholesterol biosynthesis is activated (discussed later in this chapter). The relative importance of each of these three pathways in the unfolded protein response differs in different cell types, enabling each cell type to tailor the unfolded protein response to its particular needs.
激活细胞核中基因的转录。当错误折叠的蛋白质在内质网中积累时，ATF6 蛋白被转运到高尔基体。高尔基体膜中的驻留蛋白酶切除 ATF6 的细胞质域，使其能够迁移到细胞核并帮助激活编码参与未折叠蛋白反应的蛋白质的基因转录。这种潜在膜嵌入转录因子的激活机制类似于控制胆固醇生物合成的转录调节因子的激活（在本章后面讨论）。这三条途径在未折叠蛋白反应中的相对重要性在不同细胞类型中有所不同，使每种细胞类型能够根据其特定需求调整未折叠蛋白反应。

Figure 12-37 The IRE1 limb of the unfolded protein response. Regulated RNA splicing is a key regulatory switch in the unfolded protein response pathway initiated by IRE1 (Movie 12.4). During normal conditions, IRE1 is maintained in an inactive state by its association with the ER-lumenal chaperone BiP. Elevated levels of misfolded proteins activate IRE1 by a combination of two mechanisms. First, BiP dissociates from IRE1 to bind and protect misfolded proteins from aggregation. Second, misfolded proteins bind to the lumenal domain of IRE1 facilitating the formation of IRE1 oligomers. The oligomerized IRE1 phosphorylates itself on the cytosolic side, activating its ribonuclease domain. The activated ribonuclease catalyzes the splicing of a premRNA that codes for a transcription factor that ultimately activates numerous genes in the nucleus including those coding for chaperones. Elevated chaperones help reduce the level of misfolded proteins in the ER lumen, eventually turning off IRE1 signaling.
图 12-37 IRE1 在未折叠蛋白反应中的作用。调节性 RNA 剪接是由 IRE1 启动的未折叠蛋白反应通路中的一个关键调控开关（电影 12.4）。在正常情况下，IRE1 通过与内质网腔伴侣 BiP 的结合保持在非活性状态。错误折叠蛋白的水平升高通过两种机制激活 IRE1。首先，BiP 从 IRE1 解离，以结合并保护错误折叠的蛋白质，防止其聚集。其次，错误折叠的蛋白质结合到 IRE1 的腔域，促进 IRE1 寡聚体的形成。寡聚化的 IRE1 在细胞质侧自我磷酸化，激活其核糖核酸酶域。激活的核糖核酸酶催化剪接编码转录因子的前 mRNA，最终激活细胞核中包括编码伴侣蛋白的多个基因。升高的伴侣蛋白有助于降低内质网腔中错误折叠蛋白的水平，最终关闭 IRE1 信号通路。

The ER Assembles Most Lipid Bilayers
内质网组装大多数脂质双层

Because phospholipid synthesis takes place in the cytosolic leaflet of the ER lipid bilayer, there needs to be a mechanism that transfers some of the newly formed phospholipid molecules to the lumenal leaflet of the bilayer. In synthetic lipid bilayers, lipids do not “flip-flop” in this way (see Figure 10-10). In the ER, however, phospholipids equilibrate across the membrane within minutes, which is almost 100,000 times faster than can be accounted for by spontaneous “flipflop.” This rapid trans-bilayer movement is mediated by a poorly characterized phospholipid translocator called a scramblase, which nonselectively equilibrates
由于磷脂合成发生在内质网脂质双层的细胞质叶片中，因此需要一种机制将一些新形成的磷脂分子转移到双层的腔叶片中。在合成脂质双层中，脂质并不会以这种方式“翻转”（见图 10-10）。然而，在内质网中，磷脂在膜内几分钟内达到平衡，这个速度几乎比自发“翻转”快 100,000 倍。这种快速的跨双层运动是由一种特征不明确的磷脂转运蛋白介导的，称为 scramblase，它非选择性地使磷脂达到平衡。

phospholipids between the two leaflets of the lipid bilayer (Figure 12-39). Thus, the different types of phospholipids are thought to be equally distributed between the two leaflets of the ER membrane.
脂质双层的两个叶片之间的磷脂（图 12-39）。因此，认为不同类型的磷脂在内质网膜的两个叶片之间均匀分布。

Membrane Contact Sites Between the ER and Other Organelles Facilitate Selective Lipid Transfer
内质网与其他细胞器之间的膜接触位点促进选择性脂质转移

The extensive network of the ER participates in contact sites with most other cellular organelles (Figure 12-41). As with the ER-mitochondria contact sites (see Figure 12-16), one of the main functions of these other organelle contact sites is to exchange lipids (Figure 12-42). Cells contain several families of lipid transfer proteins. Each of these can typically bind one molecule of a specific lipid (or in some cases multiple related lipids) and has additional domains that can interact with specific cellular membranes. In this manner, they serve as shuttling proteins that have distinctive specificities for the donor and acceptor membranes and the lipid they transport. Contact sites between two organellar membranes favor recruitment of the lipid transfer protein that binds these membranes, thereby enhancing the efficiency of lipid exchange. Cholesterol uses a specialized transport system from lysosomes, where it is delivered as cholesterol esters in lipoproteins, to the plasma membrane and other locations in the cell (as we discuss in Chapter 13).
内质网的广泛网络参与与大多数其他细胞器的接触位点（见图 12-41）。与内质网-线粒体接触位点（见图 12-16）一样，这些其他细胞器接触位点的主要功能之一是交换脂质（见图 12-42）。细胞中含有几类脂质转运蛋白。每种蛋白通常可以结合一个特定脂质分子（在某些情况下是多个相关脂质），并具有可以与特定细胞膜相互作用的附加结构域。通过这种方式，它们作为转运蛋白，具有对供体和受体膜及其运输的脂质的独特特异性。两个细胞器膜之间的接触位点有利于招募结合这些膜的脂质转运蛋白，从而提高脂质交换的效率。胆固醇通过一种专门的运输系统从溶酶体转运，在那里它以脂蛋白中的胆固醇酯形式被输送到质膜和细胞中的其他位置（如我们在第 13 章中讨论的）。

Summary 摘要

PEROXISOMES 过氧化物酶体

Peroxisomes are major sites of oxygen utilization and are found in virtually all eukaryotic cells. They contain oxidative enzymes, such as catalase and urate oxidase, at such high concentrations that, in some cells, the peroxisomes stand out in electron micrographs because of the presence of a crystalloid protein core (Figure 12-43). The evolutionary origin of peroxisomes is not firmly established, but they are generally thought to represent a specialized offshoot of the membrane system that composes the secretory and endocytic pathways. One hypothesis is that peroxisomes are a vestige of an ancient organelle that performed all the oxygen metabolism in the primitive ancestors of eukaryotic cells. When the oxygen produced by photosynthetic bacteria first accumulated in the atmosphere, it would have been highly toxic to most cells. Peroxisomes might have lowered the intracellular concentration of oxygen, while also exploiting its chemical reactivity to perform useful oxidation reactions. According to this view, the later development of mitochondria rendered peroxisomes less critical for cellular metabolism because many of the same biochemical reactions-which had formerly been carried out in peroxisomes without producing energy-were now coupled to ATP formation by means of oxidative phosphorylation. The oxidation reactions performed by peroxisomes in present-day cells could therefore partly be those whose functions were not taken over by mitochondria.
过氧化物酶体是氧气利用的主要场所，几乎存在于所有真核细胞中。它们含有氧化酶，如过氧化氢酶和尿酸氧化酶，浓度如此之高，以至于在某些细胞中，过氧化物酶体在电子显微镜图像中因存在结晶蛋白核心而显得特别突出（图 12-43）。过氧化物酶体的进化起源尚未确定，但通常认为它们代表了构成分泌和内吞途径的膜系统的一个专门分支。一种假说是，过氧化物酶体是一个古老细胞器的遗迹，该细胞器在真核细胞的原始祖先中执行所有的氧气代谢。当光合细菌产生的氧气首次在大气中积累时，对大多数细胞来说是高度毒性的。过氧化物酶体可能降低了细胞内的氧气浓度，同时利用其化学反应性进行有用的氧化反应。 根据这一观点，线粒体的后期发展使得过氧化物酶体在细胞代谢中的重要性降低，因为许多以前在过氧化物酶体中进行且不产生能量的生化反应，现在通过氧化磷酸化与 ATP 的形成相耦合。因此，现代细胞中由过氧化物酶体执行的氧化反应部分可能是那些未被线粒体接管的功能。

Figure 12-43 An electron micrograph of three peroxisomes in a rat liver cell. The paracrystalline, electron-dense inclusions are composed primarily of the enzyme urate oxidase. (Courtesy of Daniel S. Friend, by permission of E.L. Bearer.)
图 12-43 一张大鼠肝细胞中三个过氧化物酶体的电子显微照片。该图中的副晶体、电子密集的包涵物主要由酶尿酸氧化酶组成。（由 Daniel S. Friend 提供，经过 E.L. Bearer 的许可。）

Peroxisomes Use Molecular Oxygen and Hydrogen Peroxide to Perform Oxidation Reactions
过氧化物酶体利用分子氧和过氧化氢进行氧化反应

Short Signal Sequences Direct the Import of Proteins into Peroxisomes
短信号序列引导蛋白质进入过氧化物酶体

The proteins that compose peroxisomes are delivered by two different routes (Figure 12-46). In the first route, some of the integral membrane proteins of the peroxisomal membrane are first inserted into the ER using the ER-resident Sec61 protein translocator. These peroxisome-destined proteins are then packaged into specialized peroxisomal precursor vesicles. New precursor vesicles
组成过氧化物酶体的蛋白质通过两条不同的途径输送（图 12-46）。在第一条途径中，过氧化物酶体膜的一些整合膜蛋白首先通过内质网驻留的 Sec61 蛋白转运体插入内质网。这些目标为过氧化物酶体的蛋白质随后被包装到专门的过氧化物酶体前体囊泡中。新的前体囊泡

CCC=COCC(COP(=O)([O-])OCC[NH3+])OC(=O)CCCCC

plasmalogen 磷脂醚

Figure 12-44 The structure of a plasmalogen. Plasmalogens are very abundant in the myelin sheaths that insulate the axons of nerve cells. They make up some 80-90% of the myelin membrane phospholipids. In addition to an ethanolamine head group and a longchain fatty acid attached to the same glycerol phosphate backbone used for phospholipids, plasmalogens contain an unusual fatty alcohol that is attached through an ether linkage highlighted in yellow (bottom left).
图 12-44 磷脂醇的结构。磷脂醇在绝缘神经细胞轴突的髓鞘中非常丰富。它们占髓鞘膜磷脂的约 80-90%。除了一个乙醇胺头基和一个附着在与磷脂相同的甘油磷酸骨架上的长链脂肪酸外，磷脂醇还含有一种通过黄色高亮的醚键连接的特殊脂肪醇（左下角）。

Peroxisomal protein import is driven by ATP hydrolysis and utilizes a collection of proteins, called peroxins, that catalyze the import cycle. C-terminal peroxisomal sorting signals are recognized by the peroxin Pex5 in the cytosol. This import receptor accompanies its cargo all the way into a protein translocator in the peroxisomal membrane. After cargo release inside the peroxisome, Pex5 is recycled back to the cytosol. This recycling step requires modification of Pex5 with ubiquitin, which is used as a handle by an ATPase complex composed of Pex1 and Pex6. The Pex1-Pex6 complex harnesses the energy of ATP hydrolysis to release Pex5 from peroxisomes so it can pick up the next cargo molecule.
过氧化物酶体蛋白的进口是由 ATP 水解驱动的，并利用一组称为过氧化物酶体蛋白（peroxins）的蛋白质来催化进口循环。C 末端过氧化物酶体排序信号在细胞质中被过氧化物酶体蛋白 Pex5 识别。该进口受体将其货物一直带入过氧化物酶体膜中的蛋白质转运体。在过氧化物酶体内释放货物后，Pex5 被回收回细胞质。这个回收步骤需要用泛素修饰 Pex5，泛素作为一个把手被由 Pex1 和 Pex6 组成的 ATP 酶复合体使用。Pex1-Pex6 复合体利用 ATP 水解的能量将 Pex5 从过氧化物酶体中释放出来，以便它可以拾取下一个货物分子。

Summary 摘要

THE TRANSPORT OF PROTEINS INTO MITOCHONDRIA AND CHLOROPLASTS
蛋白质进入线粒体和叶绿体的运输

The different subcompartments in mitochondria are formed by the two concentric mitochondrial membranes (Figure 12-47A): the inner mitochondrial membrane, which encloses the matrix space and forms extensive invaginations called cristae, and the outer mitochondrial membrane, which is in contact with the cytosol. The space between the inner and outer membranes is subdivided into the crista space and intermembrane space, with protein complexes at the junctions where the cristae invaginate. Chloroplasts have an outer and inner membrane, which enclose an intermembrane space, and a stroma, which is the chloroplast equivalent of the mitochondrial matrix space (Figure 12-47B). They have an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane. The thylakoid membrane derives from the inner membrane during plastid development and is pinched off to become discontinuous with it. Each of the subcompartments in mitochondria and chloroplasts contains a distinct set of proteins.
线粒体中的不同亚区由两层同心的线粒体膜形成（图 12-47A）：内线粒体膜包围基质空间，并形成称为嵴的广泛内陷，外线粒体膜则与细胞质接触。内膜和外膜之间的空间被细分为嵴空间和膜间空间，嵴内陷的交界处有蛋白质复合物。叶绿体具有外膜和内膜，包围一个膜间空间和基质，基质是叶绿体相当于线粒体基质空间的部分（图 12-47B）。它们还有一个额外的亚区，类囊体空间，周围被类囊体膜包围。类囊体膜在质体发育过程中来源于内膜，并被挤压而与之不连续。线粒体和叶绿体中的每个亚区都包含一组独特的蛋白质。

Translocation into Mitochondria Depends on Signal Sequences and Protein Translocators
转运入线粒体依赖于信号序列和蛋白质转运体

Figure 12-47 The subcompartments of mitochondria and chloroplasts. In contrast to the cristae of mitochondria (A), the thylakoids of chloroplasts (B) are not connected to the inner membrane and therefore form a sealed continuous compartment with a separate internal space.
图 12-47 线粒体和叶绿体的亚区室。与线粒体的嵴（A）相比，叶绿体的类囊体（B）并不与内膜相连，因此形成一个密封的连续区室，具有独立的内部空间。

Figure 12-48 The protein translocators in the mitochondrial membranes. (A) The TOM, TIM, SAM, MIM, and OXA complexes are multimeric membrane protein assemblies that catalyze protein translocation across mitochondrial membranes. The protein components of the TIM22 and TIM23 complexes that line the import channel are structurally related, suggesting a common evolutionary origin of both TIM complexes. On the matrix side, the TIM23 complex is bound to a multimeric protein complex containing mitochondrial hsp70, which acts as an import ATPase, using ATP hydrolysis to pull proteins through the pore. In animal cells, subtle variations exist in the subunit composition of the translocator complexes to adapt the mitochondrial import machinery to the particular needs of specialized cell types. (B) Newly made proteins synthesized in the cytosol can use multiple routes to arrive at their final destination. The known routes via the different protein complexes are shown as green lines. There are multiple routes for a protein to become embedded into the inner and outer mitochondrial membranes, including one route for mitochondrially encoded proteins synthesized in the matrix space. SAM = sorting and assembly machinery; OXA = cytochrome oxidase activity; TIM = translocator of the inner mitochondrial membrane;
图 12-48 线粒体膜中的蛋白质转运体。(A) TOM、TIM、SAM、MIM 和 OXA 复合体是多聚体膜蛋白组装体，催化蛋白质穿越线粒体膜的转运。沿着进口通道的 TIM22 和 TIM23 复合体的蛋白质成分在结构上相关，暗示这两个 TIM 复合体具有共同的进化起源。在基质侧，TIM23 复合体与一个包含线粒体 hsp70 的多聚体蛋白复合体结合，后者作为进口 ATP 酶，利用 ATP 水解将蛋白质拉过孔道。在动物细胞中，转运体复合体的亚基组成存在细微变化，以适应线粒体进口机制对特定细胞类型的特殊需求。(B) 在细胞质中合成的新蛋白质可以通过多条途径到达其最终目的地。通过不同蛋白质复合体的已知途径以绿色线条表示。蛋白质嵌入内外线粒体膜的途径有多种，包括一种针对在线粒体基质空间合成的线粒体编码蛋白质的途径。SAM = 分拣和组装机械；OXA = 细胞色素氧化酶活性；TIM = 内线粒体膜转运蛋白；

them from the cytosol into the intermembrane space. From here, different mitochondrial proteins follow different itineraries depending on sequence features encoded in the protein. $\beta$$\beta$beta\beta-Barrel proteins, which are particularly abundant in the outer membrane, are passed to the SAM complex for insertion and folding in the outer membrane. Two different TIM complexes mediate protein transport at the inner membrane. Matrix proteins use the TIM23 complex for transport, while inner membrane proteins use the TIM22 complex, the TIM23 complex, or the OXA complex for insertion. The remainder of proteins stay in the intermembrane space where they function.
它们从细胞质转移到膜间隙。从这里，不同的线粒体蛋白根据蛋白中编码的序列特征遵循不同的路径。 $\beta$$\beta$beta\beta -桶状蛋白在外膜中特别丰富，传递给 SAM 复合体以便在外膜中插入和折叠。两种不同的 TIM 复合体介导内膜的蛋白质运输。基质蛋白使用 TIM23 复合体进行运输，而内膜蛋白则使用 TIM22 复合体、TIM23 复合体或 OXA 复合体进行插入。其余的蛋白质留在膜间隙中发挥功能。

Mitochondrial Proteins Are Imported Post-translationally as Unfolded Polypeptide Chains
线粒体蛋白质以未折叠的多肽链形式在翻译后被导入

Once the translocating protein protrudes into the intermembrane space, sequences within the polypeptide chain determine what happens next. For example, proteins destined for the matrix or inner membrane engage one of the TIM complexes and are either translocated across or inserted into the inner membrane. It is possible to rapidly cool a cell-free mitochondrial import reaction to arrest the proteins at an intermediate step during translocation. Experiments examining an arrested protein destined for the matrix show that it spans both the inner and outer mitochondrial membranes: its N -terminal signal sequence has been removed by the signal peptidase located in the matrix, while the C-terminal part of the protein is still exposed outside the mitochondria. We can therefore conclude that precursor proteins can pass through both mitochondrial membranes at once to enter the matrix space (Figure 12-49).
一旦转位蛋白突入到膜间隙，肽链内的序列决定了接下来的过程。例如，目标为基质或内膜的蛋白质会与其中一个 TIM 复合体结合，并被转位穿过或插入内膜。可以迅速冷却无细胞线粒体进口反应，以在转位过程中将蛋白质停留在一个中间步骤。研究显示，目标为基质的被停滞蛋白质跨越了内外线粒体膜：其 N 端信号序列已被位于基质中的信号肽酶去除，而蛋白质的 C 端部分仍然暴露在线粒体外部。因此，我们可以得出结论，前体蛋白可以同时穿过两个线粒体膜进入基质空间（图 12-49）。

Protein Import Is Powered by ATP Hydrolysis, a Membrane Potential, and Redox Potential
蛋白质进口依赖于 ATP 水解、膜电位和氧化还原电位

The initial use of energy, needed by most mitochondrial precursor proteins at the initial stage of the translocation process, serves to maintain the polypeptide in an unfolded state prior to import (see Figure 12-49). As discussed in Chapter 6,
大多数线粒体前体蛋白在转位过程初始阶段所需的能量初始使用，旨在保持多肽在进口前处于未折叠状态（见图 12-49）。如第六章所讨论，

(B) energy from ATP hydrolysis
（B）来自 ATP 水解的能量

© energy from redox potential
© 通过氧化还原电位获得的能量

Transport into the Inner Mitochondrial Membrane Occurs Via Several Routes
内线粒体膜的运输通过多条途径进行

In the most common translocation route, a precursor that begins in the cytosol uses the TOM and TIM23 complexes to begin import into the matrix. However, only the N -terminal signal sequence of the transported protein actually enters the matrix space (Figure 12-51A). A hydrophobic amino acid sequence, strategically located after the N -terminal signal sequence, is recognized as a transmembrane domain by the TIM23 complex. This allows insertion of the transmembrane domain into the inner membrane and prevents further translocation into the matrix, perhaps through a lateral gate analogous to that found in the ER-resident Sec61 translocator. The remainder of the protein enters the intermembrane space through the TOM complex, and the signal sequence is cleaved off in the matrix.
在最常见的转位途径中，起始于细胞质的前体通过 TOM 和 TIM23 复合体开始导入基质。然而，只有被转运蛋白的 N 端信号序列实际进入基质空间（图 12-51A）。一个位于 N 端信号序列之后的疏水氨基酸序列被 TIM23 复合体识别为跨膜域。这允许跨膜域插入内膜，并防止进一步转位进入基质，可能是通过类似于内质网驻留的 Sec61 转位体的侧向门。蛋白质的其余部分通过 TOM 复合体进入膜间隙，信号序列在基质中被切除。

The final insertion route into the inner membrane uses the OXA complex. As mentioned earlier, the OXA complex also inserts the few membrane proteins that are encoded and translated in the mitochondrial matrix (Figure 12-51C). Thus, the OXA complex can only be accessed from the matrix side of the membrane. For this reason, nuclear-encoded membrane proteins that rely on the OXA complex for insertion must first use TIM23 to translocate into the matrix (Figure 12-51D). Here, the N-terminal signal sequence is removed to expose a hydrophobic signal sequence that is then used by the OXA complex for insertion into the inner membrane.
最终插入内膜的途径使用 OXA 复合体。如前所述，OXA 复合体还插入在线粒体基质中编码和翻译的少数膜蛋白（图 12-51C）。因此，OXA 复合体只能从膜的基质侧访问。因此，依赖 OXA 复合体进行插入的核编码膜蛋白必须首先使用 TIM23 转位进入基质（图 12-51D）。在这里，去除了 N 端信号序列，以暴露出一个疏水性信号序列，然后由 OXA 复合体用于插入内膜。

Bacteria and Mitochondria Use Similar Mechanisms to Insert $\beta$$\beta$beta\beta Barrels into Their Outer Membrane
细菌和线粒体使用类似机制将 $\beta$$\beta$beta\beta 桶插入其外膜中

Two Signal Sequences Direct Proteins to the Thylakoid Membrane in Chloroplasts
两个信号序列将蛋白质导向叶绿体中的类囊体膜

The same compartments that are found in mitochondria are also in chloroplasts, and each has its distinctive complement of proteins that are selectively delivered there using mechanisms analogous to the mitochondrial systems. However, chloroplasts have an extra membrane-enclosed compartment, the thylakoid. Many chloroplast proteins, including the protein subunits of the photosynthetic system and of the ATP synthase (discussed in Chapter 14), are located in the thylakoid membrane. Many of the components of these vital complexes are encoded in the nuclear genome, and those residing in the thylakoid lumen therefore have to be imported across three membranes. The precursors of these proteins are translocated from the cytosol to their final destination in two steps using bipartite signal sequences. First, they pass across the outer and inner membranes into the stroma guided by an N -terminal chloroplast signal sequence. There, a stromal signal peptidase removes the N -terminal chloroplast signal sequence, unmasking a thylakoid signal sequence that follows it in the sequence of the precursor protein. The thylakoid signal sequence initiates integration into the thylakoid membrane or translocation into the thylakoid space (Figure 12-53A).
线粒体中发现的相同腔室也存在于叶绿体中，每个腔室都有其独特的蛋白质组成，这些蛋白质通过类似于线粒体系统的机制选择性地输送到那里。然而，叶绿体还有一个额外的膜封闭腔室，即类囊体。许多叶绿体蛋白，包括光合作用系统和 ATP 合酶的蛋白亚基（在第 14 章中讨论），位于类囊体膜中。这些重要复合体的许多组分在核基因组中编码，因此位于类囊体腔内的组分必须跨越三层膜进行进口。这些蛋白质的前体通过双部分信号序列以两步方式从细胞质转运到最终目的地。首先，它们在 N 端叶绿体信号序列的引导下，穿过外膜和内膜进入基质。在那里，基质信号肽酶去除 N 端叶绿体信号序列，暴露出紧随其后的类囊体信号序列。 类囊体信号序列启动了向类囊体膜的整合或向类囊体空间的转运（图 12-53A）。

Summary 摘要

THE TRANSPORT OF MOLECULES BETWEEN THE NUCLEUS AND THE CYTOSOL
分子在细胞核与细胞质之间的运输

The nuclear envelope encloses the DNA and defines the nuclear compartment. This envelope consists of two concentric membranes, which are perforated by nuclear pore complexes (Figure 12-54). Although the inner and outer nuclear membranes are continuous, they maintain distinct protein compositions. The inner nuclear membrane contains proteins that act as binding sites for the nuclear lamina, a meshwork of polymerized protein subunits called nuclear lamins. The lamin proteins are members of the intermediate filament family of cytoskeletal proteins (see Chapter 16). The lamina provides structural support for
核膜包围着 DNA 并定义了核区室。该膜由两层同心膜组成，膜上有核孔复合体（图 12-54）。尽管内核膜和外核膜是连续的，但它们保持着不同的蛋白质组成。内核膜含有作为核纤层结合位点的蛋白质，核纤层是由称为核纤蛋白的聚合蛋白亚单位构成的网状结构。纤蛋白是细胞骨架蛋白中间纤维家族的成员（见第 16 章）。核纤层为细胞提供结构支持。

Nuclear Pore Complexes Perforate the Nuclear Envelope
核孔复合体穿透核膜

These unstructured domains contain numerous repeats of phenylalanineglycine (FG) motifs whose weak affinity for each other creates a gel-like mesh inside the NPC. This mesh acts as a sieve that restricts the diffusion of large macromolecules while allowing smaller molecules to pass. Researchers have determined the effective size of the sieve by injecting labeled water-soluble molecules of different sizes into the cytosol and then measuring their rate of diffusion into the nucleus. Small molecules ( 5000 daltons or less) diffuse in so fast that we
这些无结构域包含大量苯丙氨酸-甘氨酸（FG）基序的重复，这些基序之间的弱亲和力在核孔复合体内形成了类似凝胶的网状结构。该网状结构充当筛子，限制大分子的扩散，同时允许小分子通过。研究人员通过将不同大小的标记水溶性分子注入细胞质，然后测量它们扩散到细胞核的速率，确定了筛子的有效大小。小分子（5000 道尔顿或更小）扩散得如此之快，以至于我们