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Chapter 15 Intracellular Compartments and Transport Essential Cell Biology Third Edition Copyright © Garland Science 2010 Hilary Truchan

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Presentation on theme: "Chapter 15 Intracellular Compartments and Transport Essential Cell Biology Third Edition Copyright © Garland Science 2010 Hilary Truchan"— Presentation transcript:

1 Chapter 15 Intracellular Compartments and Transport Essential Cell Biology Third Edition Copyright © Garland Science 2010 Hilary Truchan

2 What we see in most textbooks:

3 and what it really looks like: Figure 15-1 Essential Cell Biology (© Garland Science 2010)

4 Table 15-2 Essential Cell Biology (© Garland Science 2010) The real numbers

5 Overview Briefly describe membrane-enclosed organelles of eukaryotic cells and their functions Discuss how the protein composition of each organelle/compartment is formed and maintained Discuss how organelles communicate with each other Vesicles

6 Membrane-Enclosed Organelles

7 Figure 15-1 Essential Cell Biology (© Garland Science 2010) Internal Membranes Create Enclosed Compartments and Organelles - Segregating Metabolic Processes Examples: -Separate glycolysis from glycogenesis -Separate synthesis of protein bonds from hydrolysis of protein bonds

8 Figure 15-2 Essential Cell Biology (© Garland Science 2010) Basic set of Organelles Found in Most Animal Cells

9 Table 15-1 Essential Cell Biology (© Garland Science 2010) Functions of Organelles

10 Figure 15-3 Essential Cell Biology (© Garland Science 2010) Evolution of the ER and Nuclear Membranes Single compartment Plasma membrane invaginated forming a two- layered envelope of membrane surrounding the DNA Endomembrane System Intracellular membranes may have evolved from invagination of the plasma membrane

11 Protein Sorting

12 Cell must duplicate its membrane-enclosed organelles before dividing Most organelles formed from preexisting organelles then divide and are distributed between daughter cells Non-dividing cells continuously generate proteins and replace proteins that have been degraded Proteins need to be sorted correctly –organelle membrane proteins, organelle lumen proteins, secreted proteins –HOW? 3 Mechanisms How are proteins sorted into discrete locations?

13 1. Cytosol into the Nucleus –Nuclear pores - penetrate the inner and outer membranes –Selective gates –Transport specific molecules –Allow passive diffusion of smaller molecules Figure 15-5 Essential Cell Biology (© Garland Science 2010) How can proteins cross a phospholipid bilayer? 1. Nuclear Pores

14 2. Cytosol into ER, mitochondria, chloroplast –Transported across membrane by protein translocators - located in the membrane –Protein usually has to unfold to snake through the membrane –Similar to bacteria Figure 15-5 Essential Cell Biology (© Garland Science 2010) How can proteins cross a phospholipid bilayer? 2. Protein Translocators

15 3. ER onward and from one compartment of endomembrane system to another –Transport vesicles - loaded with cargo of proteins from the interior space of one compartment –Discharge cargo into second compartment by fusing with the membrane –Membrane components also delivered (lipids and proteins) Figure 15-5 Essential Cell Biology (© Garland Science 2010) How can proteins cross a phospholipid bilayer? 3. Transport Vesicles

16 Table 15-3 Essential Cell Biology (© Garland Science 2010) Signal Sequences - direct proteins to correct organelle Localization sequences Conserved AA sequence that acts as a molecular “address” telling the cell where this protein needs to live in the cell!

17 Figure 15-6 Essential Cell Biology (© Garland Science 2010) If localization signals are removed, the protein does not arrive at the required destination

18 How do Proteins Enter the Nucleus?

19 Figure 15-7 Essential Cell Biology (© Garland Science 2010) Nuclear envelope - defines nuclear compartment - formed from two concentric membranes Inner nuclear membrane - contain proteins that act as binding sites for the chromosomes and provide anchorage for the nuclear lamina Nuclear lamina - protein filaments that provide structural support for the nuclear envelope Outer nuclear membrane - membrane similar composition as the ER membrane (continuous with) Nuclear pores - form the gates which all molecules enter or leave the nucleus Architecture of the nucleus

20 Figure 15-8a Essential Cell Biology (© Garland Science 2010) Nuclear pores contain ~30 different proteins H 2 O filled passages - small water-soluble molecules can pass freely between nucleus and cytosol Jumble meshwork of proteins inhibit larger molecules from passing through the pore Nuclear Localization Sequence (NLS) Larger molecules need an NLS to pass through the pore - 1 or 2 short sequences of positively charged lysines or arginines Nuclear Pore Complex – A Gate

21 Figure 15-8b Essential Cell Biology (© Garland Science 2010) EM - side view of two nuclear pore complexes EM - face-on view of nuclear pore complexes EM of Nuclear Pores

22 Figure 15-9 Essential Cell Biology (© Garland Science 2010) NTR - grab onto sequences within the tangle of nuclear pore to carry the cargo into the nucleus Nuclear transport receptors actively transport proteins through nuclear pores

23 Figure Essential Cell Biology (© Garland Science 2010) Importing Proteins into the Nucleus Requires Energy - GTP hydrolysis Similar process used to carry mRNA out of the nucleus in the cytoplasm Proteins remain in fully folded conformation! Different than transport mechanisms into other parts of the cell.

24 © Sarah E Golding PhD. How do Proteins Enter the Mitochondria and Chloroplasts?

25 Figure Essential Cell Biology (© Garland Science 2010) Proteins that enter have an N-terminal signal sequence (red) Proteins translocate across both membranes simultaneously at specific sites Proteins are unfolded as they are transported Signal sequence removed after translocation completed Proteins unfold in order to enter Mitochondria and Chloroplasts HELPED BY CHAPERONES TO TRANSFER AND RE-FOLD!

26 How do Proteins Enter the Endoplasmic Reticulum?

27 Entry point for proteins destined for other organelles as well as the ER itself Proteins destined for the Golgi apparatus, lysosomes, endosomes, cell surface all first enter the ER from the cytosol Once in ER proteins do not return to the cytosol but rather travel via vesicles © Sarah E Golding PhD. Figure Essential Cell Biology (© Garland Science 2010) ER - most extensive membrane system in a eukaryotic cell

28 1.Water-soluble proteins - translocated across ER membrane into the ER lumen Destined for secretion or for the lumen of an organelle 2.Prospective transmembrane proteins - partially translocated across ER membrane and become embedded in it Destined to stay in ER membrane, membrane of another organelle, or plasma membrane Directed to ER by ER signal sequence - 8 or more hydrophobic amino acids 2 kinds of proteins transferred from the cytosol to the ER

29 Figure Essential Cell Biology (© Garland Science 2010) Proteins that enter the ER begin to enter the ER membrane before the polypeptide chain has been completely synthesized

30 Figure Essential Cell Biology (© Garland Science 2010) ER signal sequence guided to the ER membrane by: 1. A signal-recognition particle (SRP) - in the cytosol binds to the ER signal sequence when it is exposed on the ribosome 2. An SRP receptor - embedded in membrane of the ER - recognizes the SRP ER localization signals are recognized by SRPs

31 Figure Essential Cell Biology (© Garland Science 2010) ER signal sequence - usually N-terminus - functions to open channel Remains bound to channel as remainder of protein chain threaded through membrane as a large loop ER signal cleaved once proteins have crossed! Soluble proteins cross ER to enter lumen

32 How are Transmembrane Proteins Transported into the Membrane?

33 Figure Essential Cell Biology (© Garland Science 2010) Single-pass Transmembrane Proteins ER signal cleaved once proteins have crossed!

34 Double-pass Transmembrane Protein Start transfer-sequence - internal signal sequence used to start the protein transfer - never removed from peptide! Figure Essential Cell Biology (© Garland Science 2010)

35 Need additional pairs of stop and start sequences –One sequence reinitiates translocation further down peptide chain –The other stops translocation and causes polypeptide release and so on –Stitched into membrane as they are synthesized Multi-pass Transmembrane Proteins

36 Vesicular Transport

37 Two types – secretory pathway and endocytic pathway Secretory pathway –Entry into the ER is the first step - pathway to another destination –Initial destination is the Golgi apparatus –From Golgi to other compartments - carried out by budding and fusion of transport vesicles –Extend outward: ER  plasma membrane Endocytic pathway –Extend inward: plasma membrane  lysosomes –COMMUNICATION between cells! Vesicular Transport

38 Figure Essential Cell Biology (© Garland Science 2010) Secretory pathway - RED arrows Endocytic pathway - GREEN arrows Secretory and Endocytic Pathways

39 Vesicles that bud from membranes have a distinctive protein coat on cytosolic surface - coated vesicles After budding from parent organelle - sheds the coat allowing the vesicle to interact directly with the membrane it will fuse to Cells produce different types of coated vesicles – focus on Clathrin Two functions of the coat: 1.Shapes the membrane into a bud 2.Helps capture molecules for onward transport Vesicle Budding – Assembly of a Protein Coat

40 Figure 15-19b Essential Cell Biology (© Garland Science 2010) Clathrin coated vesicles watch?v=eRslV6lrVxY

41 Figure 15-19a Essential Cell Biology (© Garland Science 2010) Clathrin - best studied vesicles have coats made largely of clathrin Bud from the Golgi apparatus on the outward secretory pathway Bud from the plasma membrane on the inward endocytic pathway Electron micrograph (EM) showing a clathrin-coated vesicle forming Assemble into a basketlike network on the cytosolic surface of the membrane starts shaping the membrane into a vesicle Clathrin forms a “cage” to carry vesicles to the membrane

42 Figure Essential Cell Biology (© Garland Science 2010) Adaptins - secure clathrin coat to vesicle membrane and help select the cargo molecules Dynamin - small GTP binding protein - assembles around neck of invaginated coated pit Causes ring to contrict - pinching off vesicle from membrane Hydrolysis of GTP and help of other proteins to pinch off vesicle Clathrin coated vesicles transport select cargo molecules

43 Table 15-4 Essential Cell Biology (© Garland Science 2010) Different adaptins - adaptins that bind cargo receptors in the plasma membrane not the same as those that bind cargo receptors in the Golgi apparatus -Reflects differences in cargo molecules from each source Adaptin proteins are specific to destination

44 Actively transported by motor proteins that move along the cytoskeleton Learn More about this in Chapter 17 How are vesicles transported through the cytosol? h/research/groups- platforms/overview/unit/schoen enberger/

45 Figure Essential Cell Biology (© Garland Science 2010) Rab proteins - surface of the vesicle are recognized by tethering proteins on the cytosolic surface of the target membrane Specific combination of Rab proteins and tethering proteins - identify membrane type Ensure vesicles fuse only with the correct membrane SNAREs - transmembrane proteins important for docking the vesicle in place SNAREs on the vesicle (v-SNAREs) interact with complementary SNAREs on the target membrane (t-SNAREs) Vesicle Fusion - deliver its cargo and adds vesicle membrane to organelle Vesicle Reaches Target - Recognize and Dock with the Organelle

46 Figure Essential Cell Biology (© Garland Science 2010) Fusion requires the two lipid bilayers come within 1.5nm of each other so lipid can intermix Need to displace water from hydrophilic surface of the membrane After pairing the v-SNAREs and t-SNAREs wrap around each other = pulls two membranes into close proximity Membrane fusion sometimes needs a specific molecular signal

47 Intracellular bacteria Chlamydia spp. Mycobacterium Salmonella WHY? Reduced genome sizes compared to extracellular bacteria E. coli = 4.6 megabases Chlamydia = 1.3 megabases Do not have the genes to synthesize many essential nutrients – e.g. Amino acids Must parasitize these from their host! Take advantage of vesicular trafficking and hijack nutrient-rich vesicles!

48 Many intracellular bacteria target Rab Proteins! Mycobacterium tuberculosis acquires iron and other nutrients by targeting recycling endosome and trans- Golgi Rab Proteins Chlamydia spp. acquire sphingolipids and amino acids by targeting trans-Golgi Rab Proteins Uninfected human epithelium cells (left) with compact Golgi band close to the cell nucleus (blue) and cells infected with Chlamydiae trachomatis with Golgi fragments (red) which accumulate around the bacterial inclusion (green). Units/JuniorGroups/JRG5.html

49 How do proteins cross the plasma membrane? Secretory Pathways

50 Exocytosis - newly made proteins, lipids, and carbohydrates are delivered from the ER, via the Golgi apparatus, to the cell surface by transport vesicles that fuse with the plasma membrane FIXED sequence of membrane-enclosed compartments - often chemically modified en route Exocytosis

51 Disulfide bonds are formed between cysteine side chains Remember? Stabilize protein structure Glycosylation - covalent attachment of short oligosaccharide side chains Remember? Protect extracellular proteins, serve as transport signal form carbohydrate layer, help with cell to cell recognition –Carried out by glycosylating enzymes in ER but not in the cytosol Chemical modifications which occur in the ER

52 Figure Essential Cell Biology (© Garland Science 2010) Glycosylation - addition of oligosaccharide side chains at asparagine residues, residues added en bloc 14-sugar oligosaccharide is originally attached to specialized lipid in ER membrane – dolichol Not all proteins in ER are glycosylated. Requires specific tripeptide sequence adjacent to the modified asparagine. Glycosylation This is the beginning of protein modification! Proteins are further modified in the Golgi.

53 Proteins that function in ER - have ER retention signal Exit from ER highly selective Proteins that fold incorrectly or multi-meric proteins that fail to assemble properly are retained in ER and bind to chaperone proteins Chaperone proteins holds proteins in ER until proper folding occurs - if does not occur proteins are degraded Regulated ER Exit – protein quality control! Figure Essential Cell Biology (© Garland Science 2010)

54 Figure Essential Cell Biology (© Garland Science 2010) Unfolded protein response (UPR) - when cells protein production exceeds carrying and folding capacity UPR prompts the cell to make more ER -including all molecular machinery (lots of transcription!) UPR allows cell to adjust size of ER to met the cellular needs - if misfolded proteins continue to accumulate (out of control!) - UPR can direct cell to undergo apoptosis Unfolded protein response (UPR)

55 Oligosaccharides added in ER are further modified –removing or adding sugars Golgi apparatus Usually located near cell nucleus Collection of flattened membrane-enclosed sacs called cisternae with two distinct faces: –Cis face is adjacent to the ER –Trans face points toward the plasma membrane Outermost cisterna on each face is connected to a network of tubules and transport vesicles Proteins further modified and sorted in the Golgi apparatus

56 The Golgi apparatus! Molecular post office Proteins enter at Cis-face: move through and exit Golgi or return to ER Proteins exit at trans-face: transported to Plasma membrane or lysosomes Figure Essential Cell Biology (© Garland Science 2010)

57 Figure Essential Cell Biology (© Garland Science 2010) Secretory proteins are released by exocytosis

58 Figure Essential Cell Biology (© Garland Science 2010) Secretory cells - Regulated and Constitutive (operates continually) Pathways Constitutive secretion – all Eukaryotic cells  NO Signal Sequence Proteins incorporated into plasma membrane, extracellular matrix or are signaling molecules Regulated secretion – specialized cells  need signal to stimulate fusion with the plasma membrane and release cargo to cell exterior Example: Insulin! Constitutive Secretion vs Regulated Secretion

59 Figure Essential Cell Biology (© Garland Science 2010) Example: Release of insulin from a secretory vesicle of a pancreatic  cell Signaled to release by an increase in glucose levels in the blood Insulin is released by regulated secretion

60 How do proteins enter the cell? Endocytic Pathways Review video:

61 Endocytosis - eukaryotic cells continuously take up fluid, as well as large and small molecules Material to be ingested enclosed by a small portion of plasma membrane - buds inward and then pinches off to form an intracellular endocytic vesicle Endocytosis

62 Pinocytosis (“cellular drinking”) - ingestion of fluid and molecules via small vesicles (< 150nm in diameter) –Carried out predominantly by clathrin-coated vesicles Phagocytosis (“cellular eating”) - ingestion of large particles via large vesicles called phagosomes (generally > 250nm in diameter) –Only “phagocytic” cells do this 2 main types of Endocytosis

63 Figure Essential Cell Biology (© Garland Science 2010) Macrophage engulfing a pair of red blood cells Phagocytic cells can ingest whole cells!

64 Pinocytosis is indiscriminate Receptor-mediated Endocytosis = controlled pinocytosis –More efficient - macromolecules bind to complementary receptors on the cell surface and enter the cell as receptor-macromolecule complexes in clathrin-coated vesicles –Selective concentrating mechanism –Increases efficiency of internalization of particular macromolecules more than 1000-fold compared to pinocytosis –Example: Cholesterol Receptor-mediated Endocytosis

65 Figure Essential Cell Biology (© Garland Science 2010) LDL enters cells by receptor-mediated endocytosis

66 Two sets of endosomes –Early endosomes: Just beneath plasma membrane - mature into late endosomes as they fuse with each other –Late endosomes: Closer to the nucleus Interior of endosome kept acidic by proton pump in the endosomal membrane –pH 5-6 –Low pH causes receptors to release their cargo Main sorting station in the inward endocytic pathway –trans-Golgi is the sorting station for exocytic or secretory pathway! Endosomes – site for sorting of imported macromolecules

67 Routes taken by receptor once they enter the endosome differ according to the receptor type 1.Recycling 2.Lysosomes 3.Transcytosis Figure Essential Cell Biology (© Garland Science 2010)

68 Figure Essential Cell Biology (© Garland Science 2010) Lysosome contains hydrolytic enzymes and a proton pump Digest worn out organelles and extracellular materials pH ~5 (acidic) Unique membrane - contains transporters to allow products of the digestion to be transferred to the cytosol to be either excreted from cell or used by the cell Lysosomes are the Principal Sites of Intracellular Digestion Acidic pH and destructive enzymes are contained within membrane!

69 Figure Essential Cell Biology (© Garland Science 2010) The lysosome - The cell dumping station! Endocytosed, phagocytosed and autophagosomal material are trafficked to the lysosome for destruction! Autophagy is the destruction of an organelle – envelopes organelle and takes it to the lysosome


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