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

2. What we see in most textbooks:

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

4. The real numbers
Table Essential Cell Biology (© Garland Science 2010)

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. 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
Figure Essential Cell Biology (© Garland Science 2010)

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

9. Functions of Organelles
Table Essential Cell Biology (© Garland Science 2010)

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

11. Protein Sorting
Having briefly reviewed the main membrane-enclosed organelles of the eukaryotic cell, we turn now to the question of how each organelle acquires its unique set of proteins.

12. How are proteins sorted into discrete locations?
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

13. How can proteins cross a phospholipid bilayer?
1. Nuclear Pores
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 Essential Cell Biology (© Garland Science 2010)

14. How can proteins cross a phospholipid bilayer?
2. Protein Translocators
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 Essential Cell Biology (© Garland Science 2010)

15. How can proteins cross a phospholipid bilayer?
3. Transport Vesicles
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 Essential Cell Biology (© Garland Science 2010)

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

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

18. How do Proteins Enter the Nucleus?

19. Architecture of the nucleus
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
Figure Essential Cell Biology (© Garland Science 2010)

20. Nuclear Pore Complex – A Gate
Nuclear pores contain ~30 different proteins
H2O 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
Figure 15-8a Essential Cell Biology (© Garland Science 2010)

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

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

23. 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.
Figure Essential Cell Biology (© Garland Science 2010)

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

25. Proteins unfold in order to enter Mitochondria and Chloroplasts
HELPED BY CHAPERONES TO TRANSFER AND RE-FOLD!
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
Figure Essential Cell Biology (© Garland Science 2010)

26. How do Proteins Enter the Endoplasmic Reticulum?

27. ER - most extensive membrane system in a eukaryotic cell
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)

28. 2 kinds of proteins transferred from the cytosol to the ER
Water-soluble proteins - translocated across ER membrane into the ER lumen
Destined for secretion or for the lumen of an organelle
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

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

30. ER localization signals are recognized by SRPs
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
Figure Essential Cell Biology (© Garland Science 2010)

31. Soluble proteins cross ER to enter lumen
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!
Figure Essential Cell Biology (© Garland Science 2010)

32. How are Transmembrane Proteins Transported into the Membrane?

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

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. Multi-pass Transmembrane Proteins
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

36. Vesicular Transport

37. Two types – secretory pathway and endocytic pathway Secretory pathway
Vesicular Transport
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!

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

39. Vesicle Budding – Assembly of a Protein Coat
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:
Shapes the membrane into a bud
Helps capture molecules for onward transport

40. Clathrin coated vesicles
Figure 15-19b Essential Cell Biology (© Garland Science 2010)

41. Clathrin forms a “cage” to carry vesicles to the membrane
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
Figure 15-19a Essential Cell Biology (© Garland Science 2010)

42. Clathrin coated vesicles transport select cargo molecules
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
Figure Essential Cell Biology (© Garland Science 2010)

43. Adaptin proteins are specific to destination
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
Table Essential Cell Biology (© Garland Science 2010)

44. How are vesicles transported through the cytosol?
Actively transported by motor proteins that move along the cytoskeleton
Learn More about this in Chapter 17

45. Vesicle Reaches Target - Recognize and Dock with the Organelle
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
Figure Essential Cell Biology (© Garland Science 2010)

46. Membrane fusion sometimes needs a specific molecular signal
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
Figure Essential Cell Biology (© Garland Science 2010)

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).

49. How do proteins cross the plasma membrane? Secretory Pathways

50. Exocytosis
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

51. Chemical modifications which occur in the ER
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

52. Glycosylation
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.
This is the beginning of protein modification! Proteins are further modified in the Golgi.
Figure Essential Cell Biology (© Garland Science 2010)

53. Regulated ER Exit – protein quality control!
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
Figure Essential Cell Biology (© Garland Science 2010)

54. 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
Figure Essential Cell Biology (© Garland Science 2010)

55. Proteins further modified and sorted in the Golgi apparatus
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

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

58. Constitutive Secretion vs Regulated Secretion
Secretory cells - Regulated and Constitutive (operates continually) Pathways
Constitutive Secretion vs Regulated Secretion
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!
Figure Essential Cell Biology (© Garland Science 2010)

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

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

61. Endocytosis
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

62. 2 main types of Endocytosis
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

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

64. Receptor-mediated Endocytosis
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

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

66. Endosomes – site for sorting of imported macromolecules
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!

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

68. Lysosomes are the Principal Sites of Intracellular Digestion
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
Acidic pH and destructive enzymes are contained within membrane!
Figure Essential Cell Biology (© Garland Science 2010)

69. 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
Figure Essential Cell Biology (© Garland Science 2010)