(c) 2005 Dr. Deborah Mowshowitz , Columbia University, New York, NY. Last update 02/03/2005 02:50 PM

Handouts: 5C.RME 6A: Protein Transport

I. Wrap up of Glucose Transport from Lumen of Intestine to Body Cells. See previous lecture for details.

II. Ways that Big Molecules Enter Cells.

    A. Pinocytosis = bulk phase endocytosis; no receptor. Take in random samples of surrounding fluid. See Becker fig. 12-13. 

    B. Phagocytosis -- in specialized cells only -- extensions of cells (pseudopods) reach out and engulf solids. See Becker fig. 12-14. Vesicle that is formed is called a phagocytic vesicle (or vacuole) or phagosome. 

    C. RME = receptor mediated endocytosis. Cells take in specific substances from surrounding fluid using a receptor. See Becker fig. 12-15 (diagram) & 12-16 (micrograph). Different cell types have different combinations of receptors.

III.  RME -- Receptor Mediated Endocytosis

        A. General and/or important Features. 

1. Receptors -- Need specific receptor for each substance (or class of closely related substances) to be transported

2. Concentrates substances transported -- moves them up their gradient.

3. Requires energy (not clear exactly which stages in process use ATP or GTP.) Energy must be required because substances move against their gradients. 

4. Role of clathrin -- A peripheral membrane protein is needed to deform membrane and allow vesicles to form -- provides a coat. (See Becker figs. 12-15 to 2-18 and/or Purves 5.16.)  Other proteins are required as well, but will not be discussed.

a. Clathrin is coat protein for vesicles forming from plasma membrane and trans-Golgi. (trans side of Golgi = "far end" = side away from nucleus and ER = last part that proteins travel through as they are processed in Golgi. Also called "TGN" for "trans Golgi Network." See Purves 4.12 (5.18) or Becker fig. 12-8 for labeled pictures.)

b. Budding of other membranes involve different "coat" proteins. Best known are COPI & COPII which are involved in ER-Golgi transport. (See Becker for details if interested. Types of coats are summarized in table 12-2.) 

5. It's a cycle --  Exocytosis balances endocytosis so cell surface area stays the same. See Purves 5.15 (5.14 & 5-18) or Becker fig. 12-15. For LDL receptor, it takes about 10-20' for one "round trip." 

6. Topology -- material can enter and/or exit cell without being in contact with cytoplasm. Material can remain inside a vesicle or outside cell at all times.

B. Stages of Cycle (Numbers match steps on handout 5C.)  Click here for animation.

(1). Receptors bind material (ligand) to be internalized

(2). Receptors are in (or migrate to) coated pits (clathrin-coated parts of membrane)

(3). Membrane starts to invaginate to form coated vesicle. A single vesicle can contain more than one type of receptor plus ligand.

(4). Coated vesicle forms (pinching off of vesicle may be an energy requiring step)

(5). Uncoating occurs relatively quickly (uncoating may require energy)

(6). Vesicle is acidified to become endosome (or fuses with pre-existing endosome), and sorting of receptor(s) and ligand(s) begins.  

• A single endosome may contain many different receptors and ligands, and different ones are sorted differently. (Some examples are given in detail below.) 
• The uncoated, acidified vesicle can be called an endosome, early endosome, or a sorting vesicle. (Terminology varies -- usage of terms early endosome, late endosome etc. differs in dif. texts. ) 
• Acidification requires energy to run proton pump -- to move H⁺ into vesicle at expense of ATP. Pump is in membrane of vesicle.

Note: Details of sorting and recycling -- the remaining steps -- vary with material endocytosed. More details below for individual cases.

(7). Endosome splits.  The substance we are following, and/or its receptor, can end up in either half. (In example shown on handout, one half gets the receptor and one half gets the ligand, as is the case for LDL.) 

Note: endosome may not simply split in one step; process of sorting may be gradual. Pieces of different composition may gradually bud off as internal composition of remainder changes. 

(8). What Happens to the Different Parts of the Endosome?

8A. Fate of vesicle with materials to be recycled (receptors and/or carriers) -- this vesicle fuses with plasma membrane. (In case of LDL, this vesicle would contain the receptor for LDL.)

8B. Fate of vesicle with material that was endocytosed -- Vesicle delivers contents to appropriate cell compartment. (For LDL, delivers to  lysosomes, so material is degraded.)

(9). Exocytosis occurs -- returns receptors and/or other components to the plasma membrane or outside of cell.

   C. Some Specific Examples

        1. LDL (Low density lipoprotein) -- more details. See Becker, Box 12B or Purves 5.16 (5th ed.). 6th & 7th ed. of Purves don't have pictures, but do have info in text. Many of LDL details will be included in general case.

a. What is it? A particle containing cholesterol esters + some other lipids + a  protein (carrier).  Particle is covered by monolayer of phospholipid plus some unesterified cholesterol. 

b. Why LDL? Cholesterol is insoluble in blood. (Too hydrophobic.) Need a way to ferry cholesterol through blood and into cell -- Cholesterol transport requires formation of particle with hydrophilic surface; binding to cell surface receptor requires a protein to bind to receptor.

(1). Ligand = what actually binds receptor = protein part of LDL = carrier protein

(2). What cell actually needs is the cholesterol part.

c. Receptor, but not carrier, is recycled. Note: there are 2 separate proteins here that are easily confused 

(1) Receptor protein on the cell surface = LDL receptor = binds LDL and allows uptake of cholesterol

(2) Carrier protein  = ligand for LDL receptor = part of LDL and helps carry cholesterol through the blood. 

d. Receptor and carrier are separated inside sorting vesicles/endosomes

e. Need lysosomes to degrade carrier and release cholesterol (cholesterol esters in LDL must be split for cholesterol to be used).

(1). How LDL reaches lysosomes: vesicles/endosomes holding substrate fuse with pre-existing lysosomes, or vesicles with substrate fuse with vesicles from Golgi carrying newly made hydrolases to form new lysosomes. (More details on how hydrolases pass through the Golgi and are targeted to lysosomes to be discussed later.)

(2) Current terminology: relationship of early endosomes, late endosomes & lysosomes. Note: Most of this is FYI. In this course, the term "endosomes" will be used for both early and late endosomes.

    (a). Early endosome = sorting vesicle. Term is used differently by different authors. Can be "early" on pathway into cell (by endocytosis) and/or "early" on pathway from Golgi to lysosomes. Therefore, early endosomes can mean any of the following:

(i) (Uncoated) vesicles from invagination of plasma membrane carrying newly endocytosed material,

(ii). Vesicles coming from Golgi carrying newly made hydrolases (more on this later).

(iii) Vesicles formed by fusion of (i) + (ii). 

    (b). Late endosome = vesicle containing hydrolytic enzymes (not yet activated) plus potential substrate. More acidic than early endosome. Material not destined for lysosomes has been jettisoned.

    (c). Lysosomes = vesicle containing active hydrolytic enzymes and substrate. More acidic than late endosome. Formed by maturation of late endosome and/or fusion with pre-existing lysosome.

(3). Older terminology found in some texts (FYI only):

    (a). Primary lysosome = vesicle with enzymes only.

    (b). Secondary lysosome = enzymes + substrate = result of fusion of primary lyso. + another vesicle containing substrate.

f. Function of uptake -- to supply a nutrient (cholesterol).

2. Fe/Transferrin

a. What is transferrin? Fe needs carrier protein (like cholesterol does) for transport and binding to receptor; carrier (= ligand for cell receptor) is called transferrin

b. Both carrier & receptor are recycled

c. No lysosomes needed -- iron diffuses out of endosome; no protein is degraded

d. Carrier and receptor separate outside cell after recycled

(1). Fe/transferrin binds to receptor at neutral pH and enters cell by RME.

(2). Inside cell, Fe diffuses out of vesicle into cytoplasm, leaving apo-transferrin stuck to receptor ("apo" means without ligand or cofactor).

(3). Apo-transferrin (without Fe) sticks to receptor at low pH (in endosome) but separates at neutral pH (outside cell). This is contrary to usual behavior -- Most ligands stick to receptors at neutral pH but separate at low pH found in endosome.

(4). Note that apo-transferrin and Fe/transferrin have different affinities for the receptor at neutral pH. Under these conditions, Fe/transferrin binds to the receptor,  and apo-transferrin separates from the receptor. 

e. Function of uptake -- to supply a nutrient (Fe).

        3. EGF (Epidermal Growth Factor)

a. No carrier required; EGF (a protein -- unlike Fe or cholesterol) alone binds to receptor; EGF = signaling molecule = ligand for cell surface receptor & substance that will be transported into the cell.

b. Function of uptake -- to regulate signaling  -- turn off signal and down regulate receptors (reduce # of cell surface receptors).

c. Receptor not recycled -- Ligand (signal molecule) and receptor degraded together. 

d. Need lysosomes (to degrade both receptor and ligand)

D. For Reference: Compare & Contrast for the examples described above for transport of X

IV. Labeling -- How do you follow material coming in (or going out) of the cell? 

    A. Types of Labeling (using added tracers)

1. Continuous Labeling -- switch from regular, ordinary material to labeled material (material containing radioactivity, fluorescence, etc.) and follow what gets labeled first (with radioactivity, fluorescence, etc.), what gets labeled next, and so on. We will discuss radioactive labeling, but the principle is the same whether label is radioactivity, fluorescence, etc.

2. Pulse-Chase Experiments -- supply radioactive material for a brief time (pulse) and then switch back to ordinary, non-radioactive material (chase). Follow where the radioactivity goes. The "pulse" passes through the cell like a mouse through a boa constrictor. Just as different parts of the boa constrictor bulge out temporarily as the mouse passes down the snake, so different parts of the cell become radioactive temporarily, one at a time, as the radioactive material passes through.  Then as the "pulse" or the "mouse" passes on, each part will return to normal  -- non radioactive or normal size, depending on whether we are referring to the cell or to the snake. 

    B. Detection -- How do you find where the radioactivity is?

1. Autoradiography -- Cover a layer of labeled cells with photographic emulsion and count radioactive grains over each organelle or part of the cell. See Becker, Guide to Microscopy (or in 4th ed. the appendix, p. 831 and  figs. A-19 & A-20). This is similar to doing in situ assays, in that you examine intact cells to pin down the location of what you are looking for. 

Note: Becker's Guide to Microscopy in 5th ed. (or appendix in 4th ed) has a lot of background info on microscopic methods, including immunofluorescence, freeze fracture, etc. 

2. Fractionate First -- Break up labeled samples, fractionate into various organelles, and measure radioactivity in each fraction. This is similar to the "grind and find" procedure, in that you break up the cells, separate them into their parts, and test a solution or suspension of each part for what you are looking for. 

    C. An example: How do you follow newly made molecules moving through the cell and/or on their way out? How do we know newly made proteins go from RER to Golgi etc.?

1. General idea -- Add labeled precursors, take samples after increasing time intervals, and measure radioactivity in dif. parts of the cell by autoradiography (or measurement of radioactivity in each isolated fraction).

2. A specific example -- following secreted proteins out

a. Continuous label vs pulse-chase results (I will draw sample curves on board; see also fig. 12-10 of Becker. )

b. Implications: newly made proteins to be secreted --> RER --> Golgi --> secretory vesicles --> outside (See Becker fig. 12-8.) Click here for animation.

GFP = green fluorescent protein = small fluorescent protein made by jelly fish. Used as tag to follow proteins inside the cell. GFP is not added from outside. Instead, genetic engineering used to splice gene for GFP to gene for protein of interest. Recombinant gene makes a fusion protein = normal sequence of amino acid + GFP. Fusion protein (including GFP) is made internally by the cell; in other words, cell makes its own fluorescently tagged version of the protein. Protein usually works normally, but location of protein can be easily followed in cell,  because protein has GFP attached. GFP labeled protein is used for many purposes, including following newly made protein through the cell.

GFP is described in 6th ed. of Purves p. 324; picture of its use is on p. 885. See also the Guide to Microscopy that comes with Becker.

GFP is often used to identify cells that express (turn on) a particular gene.  For an example see http://mbclserver.rutgers.edu/driscoll/worms/gfp.jpg. The cells that "light up" are the only ones that express (turn on) the fusion gene. Only these cells produce a fusion protein containing GFP. (This example also illustrates why people use small, transparent organisms as "model organisms.") 

III. Sorting of Proteins to their Proper Place: Overview. (See handout 6A & Becker fig. 20-14 or Purves 12.14 -- terminology in Purves is slightly different.) 

    B. The Big Divide -- Attach to the ER or not? Ribosomes making protein (translating mRNA) in the cytoplasm can become attached to ER or not. How do they "decide?" Depends on what protein they make. If protein has the right "tag" (a localization signal called a signal peptide or signal sequence) the ribosomes attach to the ER. Otherwise, ribosomes remain "free" in the cytoplasm -- they do not attach to ER or any other membrane. (Note: Becker 5th ed. says there are ribosomes translating mRNA in the nucleus. Maybe, maybe not. This finding is controversial -- the existence of nuclear ribosomes is not firmly established. So we are ignoring them.)

    C. Fate of proteins made on free ribosomes

    D. Fate of proteins made on attached ribosomes -- these become part of the endomembrane system and/or leave the cell.

1. They enter the ER by co-translational import (and possibly some post-translational import). Protein can

• Remain inserted in membrane -- become a transmembrane single or multipass protein (how this works will be discussed in detail later)
• Pass all the way across the membrane and end up in lumen of ER (as soluble protein or peripheral, membrane associated protein on lumen side)

Try problem 3-1, A & B.

Next Time: How does Co-Translational Import Work? What happens inside the ER? The Golgi?