The three-dimensional structure of ubiquitin, a small protein of 76 amino acids. A family of special enzymes couples its carboxyl end to the amino group of a lysine side chain in a target protein molecule, forming an isopeptide bond. (B) Some modification patterns that have specific meanings to the cell. Note that the two types of polyubiquitylation differ in the way the ubiquitin molecules are linked together. Linkage through Lys48 signifies degradation by the proteasome (see Figure 6–87), whereas that through Lys63 has other meanings.

泛素的三维结构——一个由 76 个氨基酸构成的小蛋白。一族特殊的酶把它的羧基末端与靶蛋白分子中某个赖氨酸侧链的氨基偶联起来，形成异肽键。（B）若干对细胞具有特定含义的修饰模式。注意两类多聚泛素化在泛素分子彼此连接的方式上并不相同：通过 Lys48 连接意味着由蛋白酶体降解，而通过 Lys63 连接则另有含义。

The C-terminus of ubiquitin is initially activated by being linked via a high-energy thioester bond to a cysteine side chain on the E1 protein. This reaction requires ATP, and it proceeds via a covalent AMP–ubiquitin intermediate. The activated ubiquitin on E1, also known as the ubiquitin-activating enzyme, is then transferred to the cysteine on an E2 molecule. (B) The addition of a polyubiquitin chain to a target protein. In a mammalian cell, there are several hundred distinct E2–E3 complexes. The E2s are called ubiquitin-conjugating enzymes. The E3s are referred to as ubiquitin ligases.

泛素的 C 端首先被活化：它通过一个高能硫酯键连到 E1 蛋白的一个半胱氨酸侧链上。该反应需要 ATP，并经由一个共价的 AMP–泛素中间物进行。E1（即泛素活化酶）上被活化的泛素随后被转移到一个 E2 分子的半胱氨酸上。（B）向靶蛋白添加多聚泛素链。在哺乳动物细胞中有数百种不同的 E2–E3 复合体。E2 称为泛素结合酶，E3 称为泛素连接酶。

The 19S cap of the proteasome acts as a gate at the entrance to the inner proteolytic core, and only those proteins to be destroyed are threaded through the cap. We saw above that proteins marked for destruction are distinguished by carrying a particular type of polyubiquitin chain. This chain binds specifically to a receptor in the 19S cap of the proteasome, and here a second “check” is made by the cell to determine whether or not the protein will be destroyed. If the protein has, in addition to the ubiquitin mark, an unfolded region (which can span the mark), it is grasped tightly by the 19S cap, de-ubiquitylated, and pulled through the cap into the proteasome core.

蛋白酶体的 19S 帽子相当于内层蛋白水解核心入口处的一道闸门，只有注定要被销毁的蛋白质才会被穿过帽子送入。前文已述，被标记待降解的蛋白质是靠携带一种特定类型的多聚泛素链而被区分出来的。这条链特异性地结合到蛋白酶体 19S 帽子上的一个受体上，细胞在此进行第二次「核查」，以判定该蛋白究竟是否被销毁。如果这个蛋白除泛素标记外还带有一段未折叠区域（该区域可以跨越标记处），它就会被 19S 帽子牢牢抓住、去泛素化，并被拉过帽子进入蛋白酶体核心。

One function of intracellular proteolytic mechanisms is to recognize and eliminate misfolded or otherwise abnormal proteins, as just described. Indeed, every protein in the cell eventually accumulates damage and is probably degraded by the proteasome. Yet another function of these proteolytic pathways is to confer short lifetimes on specific normal proteins whose concentrations must change promptly with alterations in the state of a cell.

细胞内蛋白水解机制的一项功能，如前所述，是识别并清除错误折叠的或其他异常的蛋白质。事实上，细胞中每一个蛋白质最终都会累积损伤，并且很可能被蛋白酶体降解。这些蛋白水解途径的另一项功能，是赋予某些正常蛋白质以很短的寿命——这些蛋白的浓度必须随细胞状态的改变而迅速变化。

In the ER lumen, the proteins fold and oligomerize, disulfide bonds are formed, and N-linked oligosaccharides are added. The pattern of N-linked glycosylation is used to indicate the extent of protein folding, so that proteins leave the ER only when they are properly folded. Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are de-glycosylated, polyubiquitylated, and degraded in proteasomes.

在内质网腔中，蛋白质完成折叠与寡聚化，二硫键得以形成，N-连接的寡糖被添加上去。N-连接糖基化的图式被用来指示蛋白质折叠的进程，因此蛋白质只有在正确折叠之后才能离开内质网。那些不能正确折叠或寡聚化的蛋白质会被转位回细胞质基质，在那里被去糖基化、多聚泛素化，并在蛋白酶体中降解。

Like other chaperones (discussed in Chapter 6), BiP recognizes incorrectly folded proteins, as well as protein subunits that have not yet assembled into their final oligomeric complexes. It does so by binding to exposed hydrophobic amino acid sequences that would normally be buried in the interior of correctly folded or assembled polypeptide chains. The bound BiP both prevents the protein from aggregating and helps keep it in the ER (and thus out of the Golgi apparatus and later parts of the secretory pathway).

与其他分子伴侣一样（见第 6 章），BiP 能识别错误折叠的蛋白质，以及尚未装配成最终寡聚复合体的蛋白亚基。它靠结合暴露在外的疏水氨基酸序列来完成识别——这些序列在正确折叠或装配好的多肽链中本应被埋在内部。结合上去的 BiP 既能阻止蛋白聚集，又有助于把它扣留在内质网中（因而不会进入高尔基体和分泌途径的后续环节）。

Disulfide bonds stabilize the folded state of a protein, enabling it to better withstand a harsh, variable, and chaperone-free extracellular environment. Because proteins often contain multiple cysteines, they sometimes pair incorrectly. PDI resolves this problem by rearranging the disulfide bonds in a protein until it is correctly folded.

二硫键使蛋白质的折叠状态得以稳定，让它能更好地耐受严酷、多变而又没有分子伴侣保护的细胞外环境。由于蛋白质常含有多个半胱氨酸，它们有时会配对错误。蛋白二硫键异构酶（PDI）通过重排蛋白中的二硫键来解决这一问题，直到该蛋白正确折叠为止。

Many protein hormones and small neuropeptides, as well as many secreted hydrolytic enzymes, are synthesized as inactive precursors. Proteolysis is necessary to liberate the active molecules from these precursor proteins. The cleavages occur in the secretory vesicles and sometimes in the extracellular fluid after secretion. Additionally, many of the precursor proteins exit the ER with an N-terminal propeptide that is cleaved off only later in the secretory pathway to yield the mature protein.

许多蛋白类激素和小分子神经肽，以及许多分泌型水解酶，都是以无活性的前体形式合成的。必须经蛋白水解才能从这些前体蛋白中释放出有活性的分子。这些切割发生在分泌泡中，有时也发生在分泌之后的细胞外液中。此外，许多前体蛋白离开内质网时还带着一段 N 端前肽，它要到分泌途径的更后段才被切除，从而产生成熟蛋白。

The ER is organized into a netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol (Figure 12–14 and Movie 12.2). The tubules and sacs interconnect, and their membrane is continuous with the outer nuclear membrane. This membrane system encloses a single internal space, called the ER lumen, which is continuous with the space between the inner and outer nuclear membranes. The ER often occupies more than 10% of the total cell volume (see Table 12–1). The ER has a central role in the biosynthesis of both lipids and proteins, and the ER lumen stores intracellular Ca2+ that is mobilized in many cell signaling responses (discussed in Chapter 15).

内质网被组织成一个由分支小管和扁平囊构成的网状迷宫，贯穿整个细胞质基质（图 12–14）。这些小管和扁囊彼此相通，其膜与外核膜连续。该膜系统围成一个单一的内部空间，称为内质网腔，它与内、外核膜之间的间隙连续。内质网常占细胞总体积的 10% 以上。内质网在脂质和蛋白质两类物质的生物合成中都居于核心地位，内质网腔还储存细胞内的 Ca²⁺，这些 Ca²⁺ 在许多细胞信号应答中被动员释放。

The extensive ER network serves as a factory for the production of almost all of the cell’s lipids. In addition, a major portion of the cell’s protein synthesis occurs on the cytosolic surface of the rough ER: virtually all proteins destined for secretion or for the ER itself, the Golgi apparatus, the lysosomes, the endosomes, and the plasma membrane are first imported into the ER from the cytosol.

广布的内质网网络是生产细胞几乎全部脂质的工厂。此外，细胞蛋白质合成的主要部分发生在糙面内质网的细胞质面：几乎所有注定要分泌出去的蛋白，以及注定要去内质网自身、高尔基体、溶酶体、内体和细胞质膜的蛋白，都要先从细胞质基质被输入内质网。

The ER membrane is the site of synthesis of nearly all of the cell’s major classes of lipids, including both phospholipids and cholesterol, required for the production of new cell membranes. The major phospholipid made is phosphatidylcholine, which can be formed in three steps from choline, two fatty acids, and glycerol phosphate (Figure 12–38). Each step is catalyzed by enzymes in the ER membrane, which have their active sites facing the cytosol, where all of the required metabolites are found.

内质网膜是合成细胞几乎全部主要类别脂质的场所，包括建造新细胞膜所需的磷脂和胆固醇。合成量最大的磷脂是磷脂酰胆碱，它可由胆碱、两分子脂肪酸和磷酸甘油经三步反应形成（图 12–38）。每一步都由内质网膜上的酶催化，这些酶的活性部位朝向细胞质基质一侧——所需的全部代谢底物都在那里。

The main cell type in the liver, the hepatocyte, also has expanded amounts of smooth ER (see Table 12–2) serving two separate purposes. The hepatocyte is the principal site of production of lipoprotein particles, which carry lipids via the bloodstream to other parts of the body. The enzymes that synthesize the lipid components of the particles are enriched in the membrane of the smooth ER. In addition, these membranes contain enzymes that catalyze a series of reactions to detoxify drugs and various harmful compounds produced by metabolism. The most extensively studied of these detoxification reactions are carried out by the cytochrome P450 family of enzymes.

肝脏的主要细胞类型——肝细胞——也含有扩增的光面内质网，服务于两个彼此独立的目的。肝细胞是脂蛋白颗粒的主要产生部位，这些颗粒经血流把脂质运往身体其他部分；合成颗粒脂质组分的酶富集在光面内质网膜上。此外，这些膜还含有催化一系列反应的酶，用以对药物以及代谢产生的各种有害化合物进行解毒。这些解毒反应中被研究得最透彻的，是由细胞色素 P450 家族的酶完成的。

The Golgi apparatus, unlike the ER, contains many sugar nucleotides, which glycosyl transferase enzymes use to glycosylate lipid and protein molecules as they pass through the Golgi apparatus. The mannoses on the N-linked oligosaccharides that are added to proteins in the ER are often initially removed, and further sugars are added. Moreover, the Golgi apparatus is the site where O-linked glycosylation occurs and where glycosaminoglycan chains are added to core proteins to form proteoglycans. Sulfation of the sugars in proteoglycans and of selected tyrosines on proteins also occurs in a late Golgi compartment. The Golgi apparatus modifies the many proteins and lipids that it receives from the ER and then distributes them to the plasma membrane, endosomes, and secretory vesicles.

与内质网不同，高尔基体含有大量糖核苷酸，糖基转移酶利用它们对通过高尔基体的脂质和蛋白质分子进行糖基化。在内质网加到蛋白上的 N-连接寡糖，其甘露糖常常先被切除，随后再添加别的糖。此外，高尔基体是发生 O-连接糖基化的场所，也是把糖胺聚糖链加到核心蛋白上从而形成蛋白聚糖的场所。蛋白聚糖中糖的硫酸化、以及蛋白上特定酪氨酸的硫酸化，也发生在高尔基体的晚期区室。高尔基体对从内质网接收来的众多蛋白质和脂质进行修饰，然后把它们分送到细胞质膜、内体和分泌泡。

(1) Proteins with the mannose 6-phosphate (M6P) marker (see Figure 13–40) are diverted to lysosomes (via endosomes) in clathrin-coated transport vesicles. (2) Proteins with signals directing them to secretory vesicles are concentrated in such vesicles as part of a regulated secretory pathway that is present only in specialized secretory cells. (3) In unpolarized cells, a constitutive secretory pathway delivers proteins with no special features to the cell surface.

（1）带有 6-磷酸甘露糖（M6P）标记的蛋白质被装入网格蛋白包被的转运小泡，经内体转向溶酶体。（2）带有导向分泌泡信号的蛋白质在这类小泡中被浓缩，构成可调节性分泌途径，该途径只存在于特化的分泌细胞中。（3）在非极性细胞中，组成型分泌途径把不带特殊标志的蛋白质送往细胞表面。

Each Golgi stack has two distinct faces: a cis face (or entry face) and a trans face (or exit face). Both cis and trans faces are closely associated with special compartments, each composed of a network of interconnected tubular and cisternal structures: the cis Golgi network (CGN) and the trans Golgi network (TGN), respectively. The CGN is a collection of fused vesicular tubular clusters arriving from the ER. Proteins and lipids enter the cis Golgi network and exit from the trans Golgi network, bound for the cell surface or another compartment.

每一个高尔基体膜垛都有两个截然不同的面：顺面（即入口面）与反面（即出口面）。顺面和反面都各自紧邻一个特化区室，它们分别由互相连通的管状与囊状结构构成的网络组成，即顺面高尔基网（CGN）与反面高尔基网（TGN）。CGN 是从内质网来的囊泡–小管簇融合而成的集合体。蛋白质和脂质由顺面高尔基网进入，从反面高尔基网离开，去往细胞表面或另一个区室。

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 is any amino acid except proline). These sequences occur much less frequently in glycoproteins than in nonglycosylated cytosolic proteins.

多肽链几乎一进入内质网腔，就在作为靶点的天冬酰胺残基上被糖基化。前体寡糖（图中着色部分）只连接到序列 Asn-X-Ser 和 Asn-X-Thr 中的天冬酰胺侧链上（其中 X 为除脯氨酸以外的任意氨基酸）。这些序列在糖蛋白中出现的频率，远低于在不被糖基化的细胞质基质蛋白中出现的频率。

(A) Both complex oligosaccharides and high-mannose oligosaccharides share a common core region derived by trimming the original N-linked oligosaccharide added in the ER (see Figure 12–32) and typically containing two N-acetylglucosamines (GlcNAc) and three mannoses (Man). The three amino acids constitute the sequence recognized by the oligosaccharyl transferase enzyme that adds the initial oligosaccharide to the protein. Asn, asparagine; Ser, serine; Thr, threonine; X, any amino acid, except proline.

（A）复杂型寡糖与高甘露糖型寡糖共有一个核心区，它由内质网中最初加上的 N-连接寡糖经切除加工而来（见图 12–32），通常含有两个 N-乙酰葡糖胺（GlcNAc）和三个甘露糖（Man）。图中那三个氨基酸构成了寡糖转移酶所识别的序列，该酶负责把起始寡糖加到蛋白上。Asn 为天冬酰胺，Ser 为丝氨酸，Thr 为苏氨酸，X 为除脯氨酸以外的任意氨基酸。

to the hydroxyl groups of selected serines or threonines or, in some cases (such as collagens), to hydroxylated proline and lysine side chains. This O-linked glycosylation (Figure 13–33), like the extension of N-linked oligosaccharide chains, is catalyzed by a series of glycosyl transferase enzymes that use the sugar nucleotides in the lumen of the Golgi apparatus to add sugars to a protein one at a time. Usually, N-acetylgalactosamine is added first, followed by a variable number of additional sugars, ranging from just a few to 10 or more.

（有些蛋白的糖是加）到选定的丝氨酸或苏氨酸的羟基上，或在某些情况下（如胶原蛋白）加到羟基化的脯氨酸和赖氨酸侧链上。这种 O-连接糖基化（图 13–33）与 N-连接寡糖链的延伸一样，由一系列糖基转移酶催化，这些酶利用高尔基体腔内的糖核苷酸，一次一个地把糖加到蛋白上。通常先加上 N-乙酰半乳糖胺，随后再加数目可变的糖，从寥寥几个到 10 个以上不等。

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).

过氧化物酶体是氧被利用的主要场所，几乎存在于所有真核细胞中。它们所含的氧化酶类（如过氧化氢酶和尿酸氧化酶）浓度极高，以至于在某些细胞里，过氧化物酶体因存在类晶体的蛋白核心而在电镜照片中格外醒目（图 12–43）。

Peroxisomes are specialized for carrying out oxidation reactions using molecular oxygen. They generate hydrogen peroxide, which they employ for oxidative purposes—and contain catalase to destroy the excess. All peroxisomal proteins are encoded in the cell nucleus. Some of these proteins are conveyed to peroxisomes via peroxisomal precursor vesicles that bud from the ER, but most are synthesized in the cytosol and directly imported.

过氧化物酶体专司利用分子氧进行氧化反应。它们产生过氧化氢并用于氧化目的，同时含有过氧化氢酶来销毁多余的过氧化氢。所有过氧化物酶体蛋白都由细胞核基因编码。其中一部分蛋白经由从内质网出芽而来的过氧化物酶体前体小泡运送过去，但大多数是在细胞质基质中合成后被直接输入的。

Lysosomes are membrane-enclosed organelles filled with soluble hydrolytic enzymes that digest macromolecules. Lysosomes contain about 40 types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases. All are acid hydrolases; that is, hydrolases that work best at acidic pH. For optimal activity, they need to be activated by proteolytic cleavage, which also requires an acid environment. The lysosome provides this acidity, maintaining an interior pH of about 4.5–5.0.

溶酶体是被膜包被的细胞器，内部充满可溶性水解酶，用以消化大分子。溶酶体约含 40 种水解酶，包括蛋白酶、核酸酶、糖苷酶、脂肪酶、磷脂酶、磷酸酶和硫酸酯酶。它们全都是酸性水解酶，即在酸性 pH 下活性最佳的水解酶。为获得最佳活性，它们还需经蛋白水解切割而被激活，这同样要求酸性环境。溶酶体提供了这种酸度，把腔内 pH 维持在约 4.5–5.0。

About half of the soluble and membrane-bound proteins that are processed in the ER—including those destined for transport to the Golgi apparatus, lysosomes, plasma membrane, or extracellular space—are glycoproteins that are modified in this way. Some proteins in the cytosol and nucleus are also glycosylated, but not with large oligosaccharides: they instead carry a much simpler sugar modification, in which a single N-acetylglucosamine group is added to a serine or threonine of the protein.

在内质网中被加工的可溶性蛋白与膜结合蛋白中，约有一半——包括那些注定要运往高尔基体、溶酶体、细胞质膜或细胞外空间的蛋白——都是以这种方式被修饰的糖蛋白。细胞质基质和细胞核中的一些蛋白质也会被糖基化，但不是加上大的寡糖：它们携带的是一种简单得多的糖修饰，即在蛋白的某个丝氨酸或苏氨酸上加一个 N-乙酰葡糖胺基团。

N-linked glycosylation promotes protein folding in two ways. First, it has a direct role in making folding intermediates more soluble, thereby preventing their aggregation. Second, the sequential modifications of the N-linked oligosaccharide establish a “glyco-code” that marks the progression of protein folding. This glyco-code is used by chaperones and lectins in the ER to guide protein folding and degradation (discussed in Chapter 12) and by other lectins that guide ER-to-Golgi transport. As we discuss later, oligosaccharides also participate in protein sorting in the trans Golgi network.

N-连接糖基化通过两条途径促进蛋白质折叠。第一，它直接使折叠中间体更易溶解，从而防止它们聚集。第二，对 N-连接寡糖的一系列顺序修饰确立了一套「糖码」，用以标记蛋白质折叠的进程。这套糖码被内质网中的分子伴侣和凝集素用来引导蛋白质的折叠与降解（见第 12 章），也被另一些引导内质网→高尔基体转运的凝集素所使用。如后文所述，寡糖也参与反面高尔基网中的蛋白质分选。

Because chains of sugars have limited flexibility, even a small N-linked oligosaccharide protruding from the surface of a glycoprotein (Figure 13–35) can limit the approach of other macromolecules to the protein surface. In this way, for example, the presence of oligosaccharides tends to make a glycoprotein more resistant to digestion by proteolytic enzymes. It may be that the oligosaccharides on cell-surface proteins originally provided an ancestral cell with a protective coat; compared to the rigid bacterial cell wall, such a sugar coat has the advantage that it leaves the cell with the freedom to change shape and move. The sugar chains have since been adapted to serve other purposes as well. The mucus coat of lung and intestinal cells, for example, protects against many pathogens. The recognition of sugar chains by lectins in the extracellular space is important in many developmental processes and in cell–cell recognition: selectins, for example, are transmembrane lectins that function in cell–cell adhesion during blood-cell migration, as discussed in Chapter 19.

由于糖链的柔性有限，即使是从糖蛋白表面伸出的一条小小的 N-连接寡糖（图 13–35），也能限制其他大分子接近该蛋白表面。正因如此，寡糖的存在往往使糖蛋白更能抵抗蛋白水解酶的消化。有可能，细胞表面蛋白上的寡糖最初为祖先细胞提供了一层保护性外被；与坚硬的细菌细胞壁相比，这样一层糖被的好处是让细胞仍能自由改变形状和运动。此后糖链又被改造出别的用途：例如肺和肠道细胞的黏液外被可抵御多种病原体。细胞外空间中凝集素对糖链的识别，在许多发育过程和细胞–细胞识别中都很重要：例如选择素就是在血细胞迁移过程中行使细胞–细胞黏附功能的跨膜凝集素（见第 19 章）。

digestive enzymes out of the cytosol, but, even if they leak out, they can do little damage at the cytosolic pH of about 7.2. Like all other membrane-enclosed organelles, the lysosome not only contains a unique collection of enzymes but also has a unique surrounding membrane. Most of the lysosome membrane proteins, for example, are highly glycosylated, which helps to protect them from the lysosome proteases in the lumen.

（溶酶体膜把）消化酶挡在细胞质基质之外；但即便这些酶漏了出来，在约 pH 7.2 的细胞质基质环境中它们也造不成什么破坏。与其他所有膜包被细胞器一样，溶酶体不仅含有一套独特的酶，还拥有一层独特的包被膜。例如，溶酶体的大多数膜蛋白都被高度糖基化，这有助于保护它们免遭腔内溶酶体蛋白酶的降解。

Some M6P receptors, however, also take a detour to the plasma membrane, where they recapture the escaped lysosomal hydrolases and return them by receptor-mediated endocytosis (discussed later) to lysosomes via early and late endosomes. As lysosomal hydrolases require an acidic milieu to work, they can do little harm in the extracellular fluid, which usually has a neutral pH of 7.4.

不过，也有一部分 M6P 受体会绕道到细胞质膜上，在那里重新捕获逃逸出去的溶酶体水解酶，并通过受体介导的胞吞作用（后文详述）经早期和晚期内体把它们送回溶酶体。由于溶酶体水解酶需要酸性环境才能工作，它们在通常呈中性（pH 7.4）的细胞外液中造不成什么损害。

All are acid hydrolases; that is, hydrolases that work best at acidic pH. For optimal activity, they need to be activated by proteolytic cleavage, which also requires an acid environment. The lysosome provides this acidity, maintaining an interior pH of about 4.5–5.0.

它们全都是酸性水解酶，即在酸性 pH 下活性最佳的水解酶。为达到最佳活性，它们还需经蛋白水解切割而被激活，这同样要求酸性环境。溶酶体提供了这种酸度，把腔内 pH 维持在约 4.5–5.0。

In animal cells they carry a unique marker in the form of mannose 6-phosphate (M6P) groups, which are added exclusively to the N-linked oligosaccharides of these soluble lysosomal enzymes as they pass through the lumen of the cis Golgi network (Figure 13–39). Transmembrane M6P receptor proteins, which are present in the TGN, recognize the M6P groups and bind to the lysosomal hydrolases on the lumenal side of the membrane and to adaptor proteins in assembling clathrin coats on the cytosolic side. In this way, the receptors help package the hydrolases into clathrin-coated vesicles that bud from the TGN and deliver their contents to early endosomes.

在动物细胞中，它们携带一种独特的标记，即 6-磷酸甘露糖（M6P）基团；这些基团是在这些可溶性溶酶体酶通过顺面高尔基网腔时，专一地加到它们的 N-连接寡糖上的（图 13–39）。存在于 TGN 中的跨膜 M6P 受体蛋白识别 M6P 基团，在膜的腔面一侧与溶酶体水解酶结合，在细胞质一侧与正在装配的网格蛋白包被中的衔接蛋白结合。受体就这样帮助把水解酶包装进从 TGN 出芽的网格蛋白包被小泡中，并把内容物送到早期内体。

The lysosomal hydrolases contain N-linked oligosaccharides that are covalently modified in a unique way in the cis Golgi network so that their mannoses are phosphorylated. These mannose 6-phosphate (M6P) groups are recognized by an M6P receptor protein in the trans Golgi network that segregates the hydrolases and helps package them into budding transport vesicles that deliver their contents to endosomes. The M6P receptors shuttle back and forth between the trans Golgi network and the endosomes. The low pH in endosomes and the removal of the phosphate from the M6P group cause the lysosomal hydrolases to dissociate from these receptors, making the transport of the hydrolases unidirectional.

溶酶体水解酶含有 N-连接寡糖，这些寡糖在顺面高尔基网中以一种独特的方式被共价修饰，使其甘露糖被磷酸化。这些 6-磷酸甘露糖（M6P）基团被反面高尔基网中的一种 M6P 受体蛋白识别，该受体把水解酶分离出来，并帮助把它们包装进正在出芽的转运小泡，送往内体。M6P 受体在反面高尔基网与内体之间往返穿梭。内体中的低 pH，加上 M6P 基团上磷酸的去除，使溶酶体水解酶与这些受体解离，从而使水解酶的转运成为单向的。

Late endosomes containing material received from the plasma membrane by endocytosis and containing newly synthesized lysosomal hydrolases fuse with preexisting lysosomes to form structures that are referred to as endolysosomes, which then fuse with one another (Figure 13–64). When the majority of the endocytosed material within an endolysosome has been digested so that only resistant or slowly digestible residues remain, these organelles become “classical” lysosomes.

晚期内体既装有经胞吞作用从细胞质膜接收来的物质，也装有新合成的溶酶体水解酶；它们与已存在的溶酶体融合，形成被称为内体溶酶体（endolysosome）的结构，后者再彼此融合（图 13–64）。当一个内体溶酶体内大部分被胞吞的物质已被消化、只剩下难以消化或消化缓慢的残余物时，这些细胞器就成为「经典」溶酶体。

On the way to lysosomes, endocytosed material must first pass through a series of organelles called endosomes. As we will see, the ER, Golgi apparatus, lysosomes, endosomes, and plasma membrane are linked by the cell’s major pathways of membrane traffic. Mitochondria and chloroplasts generate most of the ATP that cells use to drive reactions requiring an input of free energy; chloroplasts are a specialized version of plastids (present in plants, algae, and some protozoa), which can also have other functions, such as the storage of food or pigment molecules. Finally, peroxisomes are small vesicular compartments that contain enzymes used in various oxidative reactions.

在通往溶酶体的途中，被胞吞的物质必须先经过一系列称为内体的细胞器。后文将会看到，内质网、高尔基体、溶酶体、内体和细胞质膜是由细胞的主要膜运输途径彼此连通的。线粒体和叶绿体产生细胞驱动需自由能输入的反应所用的大部分 ATP；叶绿体是质体的一种特化形式（存在于植物、藻类和某些原生动物中），质体还可有别的功能，例如贮存食物或色素分子。最后，过氧化物酶体是含有多种氧化反应用酶的小囊泡状区室。

First, the cell now has a set of internal membranes that are derived from an ancestral prokaryotic plasma membrane. These internal membranes enclose interior spaces that are said to be topologically equivalent to each other and to the exterior of the cell (Figure 12–4), because they can communicate with one another, in the sense that molecules can get from one to the other without having to cross a membrane. We shall see that this topological relationship holds for all of the organelles involved in the secretory and endocytic pathways, including the ER, Golgi apparatus, endosomes, lysosomes, and peroxisomes.

第一，细胞如今拥有一套源自祖先原核细胞质膜的内膜。这些内膜围成的内部空间被称为彼此之间、以及与细胞外部之间「拓扑等价」（图 12–4），因为它们能够互相沟通——即分子可以从一个空间到达另一个空间而无需跨越任何膜。后文将会看到，这种拓扑关系适用于参与分泌途径与胞吞途径的全部细胞器，包括内质网、高尔基体、内体、溶酶体和过氧化物酶体。

In vesicular transport, membrane-enclosed transport intermediates—which 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.

在膜泡运输中，膜包被的转运中间体——它们可以是小的球形转运小泡、拉长的小管，也可以是更大的、形状不规则的细胞器片段——把蛋白质从一个拓扑等价的区室摆渡到另一个区室。转运中间体在出芽并掐断的过程中，装载上来自起始区室腔内和膜上的货物分子。

The M6P receptor protein binds to M6P at pH 6.5–6.7 in the TGN lumen and releases it at pH 6, which is the pH in the lumen of endosomes. Thus, after the receptor is delivered, the lysosomal hydrolases dissociate from the M6P receptors, which are retrieved into transport vesicles that bud from endosomes. These vesicles are coated with retromer, a coat protein complex specialized for endosome-to-TGN transport, which returns the receptors to the TGN for reuse (Figure 13–40).

M6P 受体蛋白在 TGN 腔内 pH 6.5–6.7 的条件下结合 M6P，而在 pH 6（即内体腔的 pH）时把它释放。因此，受体送达之后，溶酶体水解酶便与 M6P 受体解离，受体则被回收进从内体出芽的转运小泡中。这些小泡由逆向运输体（retromer）——一种专司内体→TGN 运输的包被蛋白复合体——包被，把受体送回 TGN 以供重复使用（图 13–40）。

The M6P-tagged hydrolases then segregate from all other types of proteins in the TGN because adaptor proteins (not shown) in the clathrin coat bind the M6P receptors, which, in turn, bind the M6P-modified lysosomal hydrolases. The clathrin-coated vesicles bud off from the TGN, shed their coat, and fuse with early endosomes. At the lower pH of the endosome, the hydrolases dissociate from the M6P receptors, and the empty receptors are retrieved in retromercoated vesicles to the TGN for further rounds of transport. In the endosomes, the phosphate is removed from the M6P attached to the hydrolases, which may further ensure that the hydrolases do not return to the TGN with the receptor.

带 M6P 标记的水解酶随后与 TGN 中所有其他类型的蛋白分离开来，因为网格蛋白包被中的衔接蛋白（图中未示）结合 M6P 受体，而受体又结合经 M6P 修饰的溶酶体水解酶。网格蛋白包被小泡从 TGN 出芽、脱去包被，并与早期内体融合。在内体较低的 pH 下，水解酶与 M6P 受体解离，空载的受体被逆向运输体包被的小泡回收送回 TGN，进入下一轮转运。在内体中，连在水解酶上的 M6P 的磷酸被去除，这可能进一步确保水解酶不会随受体一起返回 TGN。

A vacuolar H1 ATPase in the lysosome membrane uses the energy of ATP hydrolysis to pump H+ into the lysosome, thereby maintaining the lumen at its acidic pH (Figure 13–62). The lysosome H+ pump belongs to the family of V-type ATPases and has a similar architecture to the mitochondrial and chloroplast ATP synthases (F-type ATPases), which convert the energy stored in H+ gradients into ATP (see Figure 11–12). By contrast to these enzymes, however, the vacuolar H+ ATPase exclusively works in reverse, pumping H+ into the organelle. Similar or identical V-type ATPases acidify all endocytic and exocytic organelles, including lysosomes, endosomes, some compartments of the Golgi apparatus, and many transport and secretory vesicles.

溶酶体膜上的液泡型 H⁺-ATP 酶利用 ATP 水解的能量把 H⁺ 泵入溶酶体，从而把腔内维持在酸性 pH（图 13–62）。这种溶酶体 H⁺ 泵属于 V 型 ATP 酶家族，其结构与线粒体和叶绿体的 ATP 合酶（F 型 ATP 酶）相似，后者把储存在 H⁺ 梯度中的能量转化为 ATP（见图 11–12）。但与这些酶相反，液泡型 H⁺-ATP 酶只反向工作，把 H⁺ 泵入细胞器。相似甚至相同的 V 型 ATP 酶使所有胞吞和胞吐性细胞器酸化，包括溶酶体、内体、高尔基体的某些区室，以及许多转运小泡和分泌泡。

Lysosomes are found in all eukaryotic cells. They were initially discovered by the biochemical fractionation of cell extracts; only later were they seen clearly in the electron microscope. Although extraordinarily diverse in shape and size, staining them with specific antibodies shows they are members of a single family of organelles. They can also be identified by histochemical techniques that reveal which organelles contain acid hydrolase (Figure 13–63).

溶酶体存在于所有真核细胞中。它们最初是通过对细胞抽提物做生化分级分离而被发现的，直到后来才在电子显微镜下被清楚地看到。尽管溶酶体在形状和大小上极为多样，用特异性抗体对它们进行染色表明它们属于同一个细胞器家族。它们还可以用显示哪些细胞器含酸性水解酶的组织化学技术来鉴定（图 13–63）。

These electron micrographs show two sections of a cell stained to reveal the location of acid phosphatase, a marker enzyme for lysosomes. The larger membrane-enclosed organelles, containing dense precipitates of lead phosphate, are lysosomes. Their diverse morphology reflects variations in the amount and nature of the material they are digesting. The precipitates are produced when tissue fixed with glutaraldehyde (to fix the enzyme in place) is incubated with a phosphatase substrate in the presence of lead ions.

这些电镜照片显示的是同一细胞的两个切面，经染色以显示酸性磷酸酶——溶酶体的标志酶——的定位。那些含有致密磷酸铅沉淀的较大膜包被细胞器就是溶酶体。它们形态多样，反映了所消化物质在数量和性质上的差异。这些沉淀是这样产生的：先用戊二醛固定组织（以把酶固定在原位），再在有铅离子存在的条件下与磷酸酶底物一起温育。

They contain unique membrane proteins and a wide variety of soluble hydrolytic enzymes that operate best at pH 5, which is the internal pH of lysosomes. An ATPdriven H1 pump in the lysosomal membrane maintains this low pH.

它们含有独特的膜蛋白和种类繁多的可溶性水解酶，这些酶在 pH 5 时活性最佳，而 pH 5 正是溶酶体内部的 pH。溶酶体膜上一个由 ATP 驱动的 H⁺ 泵维持着这一低 pH。

At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speed, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first vesicles and then ribosomes can be collected (Figure 8–5). All of these fractions are impure, but many of the contaminants can be removed by resuspending the pellet and repeating the centrifugation procedure several times.

在相对较低的转速下，细胞核这类大组分沉降，在离心管底部形成沉淀；转速略高时，线粒体沉淀下来；在更高的转速和更长的离心时间下，先是各种囊泡、再是核糖体可被收集下来（图 8–5）。所有这些组分都不纯，但把沉淀重悬后反复离心数次，可以除去其中许多污染物。

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).

过氧化物酶体是氧被利用的主要场所，几乎存在于所有真核细胞中。它们所含的氧化酶类（如过氧化氢酶和尿酸氧化酶）浓度极高，以至于在某些细胞里，过氧化物酶体因存在类晶体的蛋白核心而在电镜照片中格外醒目（图 12–43）。

The ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape. In this case, the sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or cesium chloride. Each cell component begins to move down the gradient as in Figure 8–6A, but it eventually reaches a position where the density of the solution is equal to its own density.

超速离心机还被用来按照细胞组分的浮力密度来分离它们，这种分离与组分的大小和形状无关。在这种情况下，样品在含极高浓度蔗糖或氯化铯的陡峭密度梯度中沉降。每一种细胞组分先像图 8–6A 那样开始沿梯度下移，但最终会到达一个位置：在那里溶液的密度恰好等于它自身的密度。

This type of oxidation reaction is particularly important in liver and kidney cells, where the peroxisomes detoxify various harmful molecules that enter the bloodstream. About 25% of the ethanol we drink is oxidized to acetaldehyde in this way. In addition, when excess H2O2 accumulates in the cell, catalase converts it to H2O through the reaction

这类氧化反应在肝细胞和肾细胞中尤为重要，那里的过氧化物酶体为进入血流的各种有害分子解毒。我们喝下的乙醇约有 25% 是以这种方式被氧化成乙醛的。此外，当细胞内积累了过量的 H₂O₂ 时，过氧化氢酶通过下面这个反应把它转化为 H₂O（即 2H₂O₂ → 2H₂O + O₂）。