Protein Trafficking 041206

Protein Trafficking – ontogeny and localization of membrane and secreted proteins

Min Li, (利民) ([email protected]) Department of Neuroscience & High Throughput Biology (HiT) Center Johns Hopkins Medical School

1

Plasmid expressing Kv channels

Transient transfection

Fixation & immunostaining

Imaging by microscopy

Why are the Kv channel proteins found on cell surface ?

Specific goals (how to get the most of the lectures)… • Understand the basic concepts • Appreciate importance and richness of biological questions and the classic experiments • Suggested Reading: Alberts et al., Chapters 10 - 14

2

Outline… • Brief review • Cells, organelles, molecules and signals • Membranes, Spatial distribution of macromolecules. • General trafficking pathways • Nuclear transport • A hallmark experiment

3

Review…information flows, integrations, outputs • • • • • • • • • • • • •

DNA Replication Transcription mechanisms and regulation DNA recombination and repair RNA Metabolism (splicing, processing, and interference) Genomics: Concepts and Applications Protein folding, Protein structure and function, DNA-protein interaction Ribosomes, translation, chemistry of post-translation modifications Cell cycle regulation Chromosome, chromatin, telomere Lipids, cell membranes and ion homeostasis Lipids, Kinases, Phosphatase, G-proteins Diffusible and electrical signaling factors Protein Trafficking

A typical cell… Size Shape Composition Weight & Density

4

Cell (size, shape, composition, & density) = f (t)

Similar architecture…different composition…thus different in function…

unstained

osmium

Nucleoside diphosphatase

Acid phosphatase

5

Two current models…

Architecture, composition, and dynamics…

6

Nerve cells (neurons) and clusters of functional molecules

Some important questions to consider… • • • • • • • •

Compartmentalization Protein partition Transport and trafficking Sorting Secretion and uptake Quality control and protein fate Protein concentration in organelles “Mis” or lack of trafficking – a common mechanism of diseases

7

Biological signal motifs in protein… • Part of a bigger structure • Confer a recognizable, but sufficiently distinct “feature” • Features – sequence, conformation, and/or chemical motifs

Signals and receptors…

8

Positions of targeting signals… ER, Periplasm

N

+ (mature) [+

Nucleus

+ + ]

+ + + + Mitochondrial Matrix

N

8 a.a. (

) (mature)

Peroxisome

Chloraplast stroma

SKL

N

OH OH OH OH OH (mature)

Questions… • Is there any evolutionary advantage to place a signal motif at a terminus? • What might be the benefits to cleave them?

9

Plasticity of signals and recognition…

Questions… • Is there any evolutionary advantage to encode “plasticity”? •

If so, what properties should plasticity have that would be more beneficial?

10

Plasticity…

11

TOPICS: • Spatial distribution of macromolecules • Nuclear Transport • Transmembrane transport • Vesicular transport

Protein expression per cell

Ghaemmaghami et al Nature 425:737

12

Subcellular distribution of proteins …

Nature Biotechnology 19, 242 - 247 (2001)

Composition of cellular membranes

13

Molecular composition of the plasma membrane … Overall: Lipids ~49%, proteins ~49% and carbohydrates~1% •

Lipids –

Phospholipids - amphipathic molecule- polar head groupnonpolar fatty acid side chains. These molecules provide the basic structure of membranes which is a bilayer (or two layers of lipids with hydrophobic side chains facing inward). The outer face is called extroplasmic and inner face is called cytoplasmic.

–

Glycolipids - found almost exclusively in the outer face of the plasma membrane, consisting of covalent linkage of a sugar molecule to a lipid. Common examples: galactocerebroside and gangliosides are prominent components of the plasma membrane of cells in the CNS.

–

Cholesterol- regulates membrane fluidity.

Molecular composition of the plasma membrane … Overall: Lipids ~49%, proteins ~49% and carbohydrates~1%

•

Proteins –

Integral membrane proteins- proteins that are embedded in the lipid bilayer

–

Peripheral proteins- proteins attached to one face or another of membrane through non-covalent interaction between lipids, integral membrane proteins or carbohydrates.

–

Functions- Proteins carry out most of the functions of membrane. They are channels, receptors, enzymes etc.

14

Molecular composition of the plasma membrane …

Overall: Lipids ~49%, proteins ~49% and carbohydrates~1%

Carbohydrates Covalently attached to proteins and lipids primarily on the exoplasmic face of the membrane. Some times observed with EM as a hazy structure called a glycocalyx.

The fluid mosaic model •

The fluid mosaic model of lipid bilayer membranes, codified by Singer and Nicolson in 1972, describes the essential features of the biological membrane. It is a two-dimensional fluid, or liquid crystal, in which the hydrophobic integral components such as lipids and membrane proteins are constrained within the plane of the membrane, but are free to diffuse laterally. Singer SJ, Nicolson GL. Science. 1972 Feb 18;175(23):720-31

15

Membrane-bound proteins … • Integral or transmembrane proteins – they have hydrophobic regions that permits sections of the protein reside with the membrane. The proteins have a cytoplasmic domain, a transmembrane domain and an extracellular domain.

• Peripheral membrane proteins – they are tethered to the membrane by other structures (not peptides) such as lipid modifications.

Plasma membrane functions…

1. Semi-permeable barrier 2. Regulate transport in and out of cell 3. Communication 4. Adhesion

16

Plasma membrane functions… 1. Semi-permeable barrier: The lipid bilayer accounts for the basic barrier functions of the plasma membrane. • •

permeable to water, O2 and small hydrophobic molecules such as steroids, ethanol impermeable highly charged molecules charged ions such as Na, K.

2. Regulate transport in and out of cell. 3. Communication 4. Adhesion

Plasma membrane functions… Plasma membrane functions 1.

Semi-permeable barrier:

2. Regulate transport in and out of cell. Transport in Endocytosis - uptake of substances outside of the cell via invaginations in the plasma membrane that ultimately pinch off and form a membrane-bound body called an endosome. Pinocytosis - uptake of fluid or small molecules in vesicles less than 50nm in diameter. Phagocytosis - uptake of large particles > than 250nm (i.e. bacteria by macrophages) Potocytosis - uptake of small molecules through small caveolin lined pits that have a localized concentration of receptors. These invaginations do not pinch off to become vesicles Receptor Mediated Endocytosis - Binding of a ligand to a receptor triggers endocytosis through a clathrin-coated pit. Clathrin is a fuzzy protein on coated pit and this protein mediates this process. Uptake of Iron LDL EGF. Transcytosis - movement of molecules in caveolin-lined pits from one surface of the cell membrane to another. (Important for IGA transport across the small intestine)

Transport out Exocytosis - (secretion): fusion of a membrane-bound vesicle with the plasma membrane, resulting in the discharge of the internal contents into extacellular space. constitutive secretion (bulk flow secretion) - constant fusion of vesicles with plasma membrane. Most proteins on membrane get here this way. regulated secretory pathway - secretory vesicles require a signal to fuse with plasma membrane i.e., neurotransmitter release from nerve cells. Coordination - endocytosis and exocytosis are coordinately regulated to maintain plasma membrane size

3. Communication 4. Adhesion

17

Plasma membrane functions… Plasma membrane functions 1. Semi-permeable barrier: 2. Regulate transport in and out of cell. 3. Communication Pathways Endocrine - communication at a distance i.e. hormones produced by endocrine organ signaling a target tissue Paracrine - local communication between different cell types within a tissue Autocrine - local communication between the same cell type within a tissue

Molecules Gas based - (CO, NO) pass through plasma membrane, bind directly to an enzymatic signal transducer in cytoplasm Hydrophobic molecules - (steroids , retinoids) pass through plasma membrane, bind receptor in cytoplasm, receptor binds directly to DNA; can influence gene expression. Hydrophilic molecules - (peptide growth hormones, neurotransmitters) cannot cross membrane, bind to receptor (protein) located on external surface of membrane.

4. Adhesion

Plasma membrane functions… Plasma membrane functions 1. Semi-permeable barrier: 2. Regulate transport in and out of cell. 3. Communication 4. Adhesion Functions Holds tissues together Cell mobility Adhesion-related communication determines cell fate Immune system function - cell adhesion processes vital to the cell-cell recognition that occurs between cells of the immune system. Terms related to adhesion Adhesion molecules - actual molecules that hold a cell to another or a substrate Homotypic - binding between adhesion molecules that are of the same type. Heterotypic - binding between adhesion molecules that are of different types. Types of adhesion molecules

18

TOPICS: • Spatial distribution of macromolecules • Nuclear Transport • Transmembrane transport • Vesicular transport

Solutions of protein partition…

19

Asymmetric “cycling”…how?

I. Gated transport - nuclear transport (enter and exit) …

20

Gated transport - nuclear transport (enter and exit) … • The nuclear pore is the site of transport • The substrate to be transported has size exclusion preference

Experiments:

• The soluble factors that are required for proper movement of a particular substrate

Particles up to 9 nm in diameter (corresponding to globular proteins up to 40 kDa) can pass through NPCs by passive diffusion.

21

The Nuclear Pore …organization • 100 nM in diameter • 8-fold rotationally symmetric • Asymmetric with regard to the nuclear vs. cytoplasmic side • Composed of ~ 60-100 proteins that termed nucleoporins. Each NPC in human cells has a mass of ~120 x 106 daltons, which is 40 times that of a ribosome. • Some nucleoporins contains characteristic amino acid repeats.

The Nuclear Pore …organization • 100 nM in diameter • 8-fold rotationally symmetric • Asymmetric with regard to the nuclear vs. cytoplasmic side • Composed of ~ 60-100 proteins that termed nucleoporins. Each NPC in human cells has a mass of ~120 x 106 daltons, which is 40 times that of a ribosome. • Some nucleoporins contains characteristic amino acid repeats.

22

Asymmetric “cycling”…how?

How would the directional transport across the NPC occurs?)

The Nuclear Pore …selectivity Signals – Nuclear Localization Motifs (NLS) • Abundant nucleoplamin protein showed that C-terminus was required – could not be explained by diffusion and/or retention inside the nucleus. • “K(K/R)X(K/R) “ of SV40 Large T-antigen is until recently considered the “canonical” NLS. Necessary and sufficient for mediating the transport. • Other sequences of NLS activity have now been identified – e.g., M9 sequence from hnRNPA1 and others. • Evidence – for existence of a receptor?

23

The Nuclear Pore …selectivity Gating Factors: •

The first NLS receptor is heterodimer with several names (Importin, karyopherin, NLS receptor and SRP1 (not to be confused with the one involved in ER translocation!)]. Two subunits: • alpha: 65 KDa - binds to NLSs • beta: 90 KDa – mediates docking at the pore

•

There are other signals target proteins for nuclear export (Protein are made in cytoplasmic compartment, Why there is a need for nuclear export instead of diffusion?). One class is leucine-rich and termed NES (nuclear export signal). NESs are recognized by a member of importin family – termed Exportin or Crm1.

•

Another key protein involved is a small GTPase – known as Ran. It is required for translocation of the importin-substrate complex either into or out of the nucleus. All known Importins and Exportins appear to bind Ran.

•

The mRNAs, an export machinery is suggested and distinct from the protein export machinery.

Ran – mediated “cycling”…

24

Ran – mediated “cycling”…

How would the directional transport across the NPC occurs?

mRNA transport into cytosol…

25

Asymmetric “cycling”…how?

Solutions of protein partition… 1.

Eukaryotic cells have an elaborate system of internal membrane-bound structures called organelles.

2.

Each organelle has a unique composition of (glyco)proteins and (glyco)lipids that carry out a particular set of functions.

3.

An organelle comprises one or more membrane-bound compartments.

4.

Organelles may act autonomously or in cooperation to accomplish a given function.

5.

In the endocytic and exocytic pathways, cargo proteins are transferred between compartments by transport vesicles that form by budding from an organelle's surface.

6.

Transport vesicles can selectively include material destined for transfer and exclude material that must remain in the organelle from which they bud.

7.

Selective inclusion into transport vesicles is ensured by signals in a protein's amino acid sequence or carbohydrate structures.

8.

Transport vesicles contain proteins that target them specifically to their intended destinations.

26

What force(s) drive a protein to cell surface?

pH Gradient Membrane Thickness Bulk Flow

Feldherr CM, J Cell Biol. 1969 Sep;42(3):841-5.

27

Feldherr CM, J Cell Biol. 1969 Sep;42(3):841-5.

Feldherr CM, J Cell Biol. 1969 Sep;42(3):841-5.

28

Feldherr CM, J Cell Biol. 1969 Sep;42(3):841-5.

29