Cell Structure and Function

Structure of the Nuclear envelope

Nuclear envelope - composed of: 1. inner and outer membranes 2. nuclear lamina 3. nuclear pore complex
Nuclear lamina - A meshwork of lamin filaments providing structural support to the nucleus -serve as a site of chromatin attachment

Nuclear pore complex -A large structure forming a transport channel through the nuclear envelope.

Lamins. Most mammalian cells, for example, contain four different lamins, designated A, B1, B2, and C. All the lamins are 60- to 80-kilodalton (kd) fibrous proteins that are related to the intermediate filament proteins of the cytoskeleton

Molecular traffic through nuclear pore complexes

Model of the nuclear pore complex

Selective Transport of Proteins to and from the Nucleus

Proteins are targeted to the nucleus by specific amino acid sequences, called nuclear localization signals

Protein import through the nuclear pore complex

- can be: 1. energy-requiring process 2. does not require energy

A. Non-energy requiring step

-proteins that contain nuclear localization signals bind to the nuclear pore complex but do not pass through the pore nuclear localization signals

recognized by a cytosolic receptor protein importin

receptor-substrate complex binds to the nuclear pore.

Importin
consists of two subunits: 1. importin binds to the basic amino acid-rich nuclear localization signals of proteins such as T antigen and nucleoplasmin. 2. importin binds to the cytoplasmic filaments of the nuclear pore complex, bringing the target protein to the nuclear pore. :

Table 12-3. Some Typical Signal Sequences

B. Energy-requiring step -requires GTP hydrolysis. A key player in the translocation process is a small GTP-binding protein called Ran, (involved in nuclear import and export)

Enzymes that stimulate GTP binding to Ran are localized to the nuclear side of the nuclear envelope whereas enzymes that stimulate GTP hydrolysis are localized to the cytoplasmic side. Role of the Ran protein in nuclear import 1.Transport through the nuclear pore complex is driven by a gradient of Ran/GTP, - a high concentration of Ran/GDP in the cytoplasm - a high concentration of Ran/GTP in the nucleus.
2.Complexes form between target proteins containing nuclear localization signals (NLS) importin , and importin in the cytoplasm where Ran is in the GDP-bound form.

3. Transport through the nuclear pore complex, Ran/GTP binds to importin , releasing importin and the target protein in the nucleus.

4. The Ran/GTP-importin complex is then transported back to the cytoplasm, where the Ran GTPase-activating protein (Ran GAP) stimulates hydrolysis of the bound GTP to form Ran/GDP.

5. This conversion of Ran/ GTP to Ran/GDP is accompanied by release of importin .

Role of the Ran protein in nuclear import

Nuclear export

Proteins are targeted for export from the nucleus by specific amino acid sequences, called nuclear export signals.

*the nuclear export receptors (called exportins) are related to importin 1. Complexes between target proteins bearing nuclear export signals (NES), exportins, and Ran/ GTP form in the nucleus. 2. transport through the nuclear pore complex, Ran GAP stimulates the hydrolysis of bound GTP, leading to formation of Ran/GDP and release of the target protein and exportin in the cytoplasm.

3. Exportin is then transported back to the nucleus.

The Nucleolus
the most prominent substructure within the nucleus ribosome production factory, designed to fulfill the need for large-scale production of rRNAs and assembly of the ribosomal subunits. morphologically, nucleoli consist of three distinguishable regions: the fibrillar center, dense fibrillar component, and granular component

not surrounded by a membrane

organized around the chromosomal regions that contain the genes for the 5.8S, 18S, and 28S rRNAs.

The Nucleus during Mitosis

1. Dissolution of the Nuclear Envelope
-the disassembly of the nuclear envelope marks the end of the prophase of mitosis (not a universal feature of mitosis and does not occur in all cells) a. Closed mitosis - in which the nuclear envelope remains intact - the daughter chromosomes migrate to opposite poles of the nucleus, which then divides in two. - occurs in some unicellular eukaryotes (e.g., yeasts) b. Open mitosis - characterized by breakdown of the nuclear envelope. - daughter chromosomes then migrate to opposite poles of the mitotic spindle, and new nuclei reassemble around them - occur in cells of higher eukaryotes

Disassembly of the nuclear envelope

-involves changes in all three of its components: 1. the nuclear membranes are fragmented into vesicles, 2. the nuclear pore complexes dissociate, 3. and the nuclear lamina depolymerizes.

At mitosis, Cdc2 and other protein kinases phosphorylate the lamins, causing the filaments to dissociate into free lamin dimers.

2. Chromosome Condensation
-Recent studies have also identified protein complexes called condensins that play a major role in chromosome condensation. -Condensins are phosphorylated and activated by the Cdc2 protein kinase, providing a direct link between activation of Cdc2 and mitotic chromosome condensation

3. Re-formation of the Interphase Nucleus

-Chromosome decondensation and reassembly of the nuclear envelope appear to be signaled by inactivation of Cdc2, which was responsible for initiating mitosis by phosphorylating cellular target proteins, including the lamins, histone H3, and condensins.

-Inactivation of Cdc2 leads to the dephosphorylation of the proteins that were phosphorylated at the initiation of mitosis, resulting in exit from mitosis and the re-formation of interphase nuclei.

In concert with dissolution of the nuclear lamina, the nuclear membrane fragments into vesicles

As the nuclear lamina dissociates, the nuclear membrane fragments into vesicles. The B-type lamins remain bound to these vesicles, while lamins A and C are released as free dimers.

Re-formation of the nuclear envelope

1. The first step in reassembly of the nuclear envelope is the binding of membrane vesicles to chromosomes, which may be mediated by both integral membrane proteins and Btype lamins. The vesicles then fuse, the nuclear lamina reassembles, and the chromosomes decondense

2. 3. 4.

All Eucaryotic Cells Have the Same Basic Set of Membraneenclosed Organelles

Figure 12-1. The major intracellular compartments of an animal cell.

Figure 17-1. Overview of sorting of nuclear-encoded proteins in eukaryotic cells

Endoplasmic reticulum (ER).

-network of membrane-enclosed tubules and sacs (cisternae) that extends from the nuclear membrane throughout the cytoplasm -largest organelle of most eukaryotic cells -functions as a store for Ca2+ ions.

2 types: 1. rough ER-ribosomes on its outer surface, functions in protein processing - site of synthesis of proteins destined for secretion 2. smooth ER-not associated with ribosomes and is involved in lipid, rather than protein, metabolism

The Endoplasmic Reticulum and Protein Secretion

These experiments defined a pathway taken by secreted proteins, the secretory pathway: rough ER Golgi secretory vesicles cell exterior.

The secretory pathway (Pancreatic acinar cells )

Two types of Protein translocation in cell

1. Cotranslational translocation - Proteins can be translocated into the ER either during their synthesis on membrane-bound ribosomes (eg. mammalian cells) 2. Posttranslational translocation- Proteins can be translocated into the ER after their translation has been completed on free ribosomes in the cytosol

* both cotranslational and posttranslational pathways are used in yeast.

Signal sequence
A hydrophobic sequence at the amino terminus of a polypeptide chain that targets it for secretion in bacteria or incorporation into the endoplasmic reticulum in eukaryotic cells. *signal recognition particle (SRP)

Cotranslational targeting of secretory proteins to the ER

Posttranslational translocation of proteins into the ER

Proteins destined for incorporation into the plasma membrane or the membranes of the ER, Golgi, or lysosomes are initially inserted into the ER membrane instead of being released into the lumen.

ER is also the 1. site of protein folding, 2. assembly of multisubunit proteins, 3. disulfide bond formation, 4. the initial stages of glycosylation, 5. and the addition of glycolipid anchors to some plasma membrane proteins. *Indeed, the primary role of lumenal ER proteins is to catalyze the folding and assembly of newly translocated polypeptides.

The Smooth ER and Lipid Synthesis

-major site at which membrane lipids are synthesized in eukaryotic cells. -synthesized lipids are transported from the ER to their ultimate destinations either in vesicles or by carrier proteins -abundant in cell types that are particularly active in lipid metabolism (eg. steroid-producing cells, such as those in the testis and ovary; liver, where it contains enzymes that metabolize various lipid-soluble compounds

flippases
membrane proteins catalyze the rapid translocation of phospholipids across the ER membrane, resulting in even growth of both halves of the bilayer.

Golgi apparatus
-consists of organized stacks of disclike compartments called Golgi cisternae; it receives lipids and proteins from the ER and dispatches them to a variety of destinations, usually covalently modifying them en route
Regions of the Golgi apparatus Vesicles from the ER fuse to form the ER-Golgi intermediate compartment, and proteins from the ER are then transported to the cis Golgi network. Resident ER proteins are returned from the ER-Golgi intermediate compartment and the cis Golgi network via the recycling pathway. The medial and trans compartments of the Golgi stack correspond to the cisternae in the middle of the Golgi complex and are the sites of most protein modifications. Proteins are then carried to the trans Golgi network, where they are sorted for transport to the plasma membrane, secretion, or lysosomes.

-In addition to its activities in processing and sorting glycoproteins, the Golgi apparatus functions in lipid metabolismin particular, in the synthesis of glycolipids and sphingomyelin -In plant cells, the Golgi apparatus has the additional task of serving as the site where complex polysaccharides of the cell wall are synthesized (eg. hemicellulose and pectins)

Protein Sorting and Export from the Golgi Apparatus

-involves the sorting of proteins into different kinds of transport vesicles, which bud from the trans Golgi network and deliver their contents to the appropriate cellular locations Types of secretory pathway: 1. constitutive secretory pathway some proteins are carried from the Golgi to the plasma membrane -accounts for the incorporation of new proteins and lipids into the plasma membrane, as well as for the continuous secretion of proteins from the cell. 2. distinct pathway of regulated secretion - Other proteins are transported to the cell surface

3. specifically targeted to other intracellular destinations - such as lysosomes in animal cells or vacuoles in yeast.

Transport from the Golgi apparatus

In yeasts and plant cells, which lack lysosomes, proteins are transported from the Golgi apparatus to an additional destination: the vacuole -assume the functions of lysosomes in these cells as well as performing a variety of other tasks, such as the storage of nutrients and the maintenance of turgor pressure and osmotic balance.

Lysosomes
-contain digestive enzymes that degrade defunct intracellular organelles, as well as macromolecules and particles taken in from outside the cell by endocytosis. On their way to lysosomes, endocytosed material must first pass through a series of organelles called endosomes.

The intracellular compartments of the eucaryotic cell involved in the biosynthetic- secretory and endocytic pathways

Vesicular Transport
-a major cellular activity, responsible for molecular traffic between a variety of specific membrane-enclosed compartments. -understanding the molecular mechanisms that control vesicle packaging, budding, and fusion is being considered

Coat Proteins and Vesicle Budding

-first step in vesicular transport is the formation of a vesicle by budding from the membrane. -cytoplasmic surfaces of transport vesicles are coated with proteins, and it appears to be the assembly of these protein coats that drives vesicle budding by distorting membrane conformation Three kinds of coated vesicles, which appear to function in different types of vesicular transport: 1. clathrin-coated vesicles -responsible for the uptake of extracellular molecules from the plasma membrane by endocytosis as well as the transport of molecules from the trans Golgi network to lysosomes

2. nonclathrin-coated or COP-coated vesicles (COP stands for coat protein) - coated vesicles identified as budding from the ER and Golgi complex 2 classes: 1. COPII-coated vesicles - bud from the ER and carry their cargo forward along the secretory pathway, to the Golgi apparatus.
2. COPI-coated vesicles - bud from the ER-Golgi intermediate compartment or the Golgi apparatus and function in the retrieval pathways that serve to retain resident proteins in the Golgi and ER.

Incorporation of lysosomal proteins into clathrin-coated vesicles

Vesicle Fusion
-transport vesicle with its target involves two types of events: 1. transport vesicle must specifically recognize the correct target membrane 2. the vesicle and target membranes must fuse, thereby delivering the contents of the vesicle to the target organelle.

vesicle fusion is mediated by interactions between specific pairs of proteins, called SNAREs, on the vesicle and target membranes (v-SNAREs and t-SNAREs, respectively)

Rab proteins are a family of small GTP-binding proteins that are related to the Ras proteins

Vesicle fusion - mediated by binding between specific pairs of v-SNAREs and t-SNAREs on the vesicle and target membranes, respectively. Rab GTP-binding proteins are required to facilitate formation of v-SNARE/t-SNARE complexes. Following membrane fusion, the NSF/SNAP proteins disassemble the SNARE complex.

The lysosome system

Organization of the lysosome

Endocyto sis and lysosome formation

Peroxisomes
- are small vesicular compartments that contain enzymes utilized in a variety of oxidative reactions.

Mitochondria and (in plants) chloroplasts generate most of

the ATP used by cells to drive reactions that require an input of free energy; chloroplasts are a specialized version of plastids, which can also have other functions in plant cells, such as the storage of food or pigment molecules