Biological Membranes - A. N. Ogurtsov 2012

Structure and Functions of Biomembranes
Intracellular Protein Transport
Vesicular Transport

Glycoproteins transported from the rough Endoplasmic reticulum to other destinations are "packaged" into small transport vesicles that bud off from the transitional region of The endoplasmic reticulum. The walls of these vesicles consist of Lipids and Membrane Proteins of the reticulum, while the vesicles themselves contain Soluble Glycoproteins captured from the lumen of the endoplasmic reticulum. Most of these vesicles fuse to form flat cisternae on the side of the Golgi apparatus facing The Nucleus, known as the forming or cis-face (forming pole).

Within the cisternae of the Golgi apparatus, further "maturation" of the proteins synthesized in the rough Endoplasmic reticulum and transferred to the Golgi apparatus takes place.

Protein "maturation" involves various covalent modifications through which proteins acquire their functionally active Structure. In addition, Oligosaccharides linked to asparagine residues, previously attached to proteins in the endoplasmic reticulum, undergo extensive modification: certain sugar residues are selectively cleaved off, while new ones are added.

Oligosaccharide Processing is carried out by several distinct, highly complex, and precisely "programmed" enzymatic mechanisms. The choice of the modification "program" is determined by some (as yet unknown) property of each individual polypeptide chain.

The Enzymes that perform protein modification, much like the modifying proteins themselves, likely reach the Golgi apparatus from the endoplasmic reticulum. It is currently believed that, following processing, the oligosaccharide attached to the protein serves as an "address" indicating where the given protein must be delivered.

The "matured" proteins are "packaged" once again into transport vesicles, but this time at the mature or trans-face (secretory pole).

A GENERALIZED SCHEME OF vesicular transport is presented in Figure 83. Proteins synthesized in the rough ER (Figure 83(1)) are packaged into transport vesicles (Figure 83(2)), which bud off from the RER and fuse together to form a new cis-Golgi network (CGN) cisterna.

Enzymes and Proteins of the endoplasmic reticulum that ended up in such a vesicle, as well as membrane proteins responsible for vesicle formation, are returned to the rough endoplasmic reticulum for "reuse" (Figure 83(3)) via vesicles that bud off from the cis-face of the Golgi apparatus and fuse with the membrane of the rough endoplasmic reticulum.

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Figure 83 - Generalized scheme of vesicular transport

Each Golgi cisterna from the cis-region physically moves through the intermediate region into the trans-region of the Golgi apparatus via a non-vesicular process known as cisternal progression (Figure 83(4)).

The "retrieval" of region-specific proteins of the Golgi apparatus is carried out by a vesicular mechanism (Figure 83(5)). In all Cells, certain proteins constantly move toward the surface in vesicles that have budded off from the trans-face of the Golgi apparatus, ensuring continuous protein secretion outside The Cell (constitutive exocytosis) (Figure 83(6)). In certain cell types, specific proteins are "stored" in secretory vesicles (Figure 83(7)) and are secreted only upon receiving an external neural or hormonal signal (regulated exocytosis).

Proteins and lipids destined for Lysosomes (Figure 83(8)) bud off from the trans-face of the Golgi apparatus and join secondary endosomes, which subsequently fuse with the lysosome. Proteins from the extracellular space and Plasma Membrane proteins that form endocytic vesicles during endocytosis (Figure 83(9)) (pinching off from The Plasma Membrane into the cell's interior) are likewise transported into lysosomes via endosomes.

Most transport vesicles represent a special class of intracellular vesicles that appear on microphotographs to be surrounded by a bristle-like coat on the cytoplasmic side, and are therefore called coated vesicles. The diameter of coated vesicles ranges from 50 to 250 nm.

The coat is formed by Three types of proteins:

1) COPII protein coats vesicles transporting proteins from the rough endoplasmic reticulum to the Golgi apparatus;

2) COPI protein coats vesicles carrying out "retrieval"

transport of proteins between Golgi cisternae and back to the rough endoplasmic reticulum;

3) clathrin coats vesicles that transport proteins from the plasma membrane and the trans-Golgi network to secondary endosomes (Figure 84).

Figure 84 - STRUCTURE OF THE clathrin coat: a - triskelion; b - packaging of triskelions into the clathrin shell; c - clathrin cages formed by spontaneous aggregation of triskelions in vitro; 1 - clathrin; 2 - small polypeptide; 3 - clathrin binding site upon aggregation

Clathrin (180 kDa), together with a smaller polypeptide (35 kDa), forms a characteristic polyhedral shell On the surface of coated vesicles.

The main structural element of the shell is the protein complex known as a triskelion, consisting of three clathrin polypeptide chains and three small (35 kDa) Polypeptides.

Triskelions form basket-like lattices of hexagons and pentagons on The surface of coated vesicles.

Isolated triskelions are capable of spontaneous aggregation under appropriate conditions. In this process, typical polyhedral cages are formed even in the absence of vesicles (Figure 84(b)).

A multitude of accessory proteins are integrated into the vesicle membranes. These proteins, firstly, "fish out" proteins with identical oligosaccharide "addresses" from the Golgi cisternae during vesicle formation, and secondly, recognize the Components of the membrane with which the given vesicle must fuse (Figure 85).

Figure 85 - Scheme of vesicular processes: a - formation of a coated vesicle from the donor membrane; b - fusion of the vesicle with the target membrane; 1 - donor membrane; 2 - Water-soluble transported protein; 3 - GTP-binding protein; 4 - v-SNARE protein; 5 - membrane-integrated transported protein; 6 - membrane receptor for water-soluble transported proteins; 7 - coat proteins; 8 - target membrane; 9 - t-SNARE proteins; 10 - t-SNARE complex

It is known that the contents of each transport vesicle are delivered precisely to the correct "address" at a specific intracellular membrane; therefore, there must exist distinct subpopulations of transport vesicles bearing unique accessory proteins on their surface, the so-called docking markers (v-SNARE proteins) (Figures 85 and 86), which are recognized by complementary acceptors (t-SNARE proteins) on target membranes to form the SNARE complex.

The coat plays a key role in vesicle formation, but shortly after the vesicle buds off, the coat proteins dissociate from its surface (uncoating), and the absence of the coat allows the vesicle to fuse with the membrane at the end of transport.

The scheme of vesicular transport between the rER and the cis-Golgi is shown in Figure 86. Forward (anterograde) transport is mediated by COPII-coated vesicles (Figure 86(1,2,3)), which are formed by the polymerization of COPII proteins on the surface of the endoplasmic reticulum membrane.

Figure 86 - Vesicular protein transport between the endoplasmic reticulum and the cis-Golgi region

Dissociation of COPI proteins from the vesicle surface exposes v-SNARE proteins. The pairing of v-SNARE with t-SNARE proteins on the surface of the cis-Golgi network forms SNARE complexes, thereby enabling membrane fusion.

Return (retrograde) transport (Figure 86(4,5,6)), carried out via vesicle coating with COPI proteins, ensures the retrieval (recycling) of Membrane Lipids, specific proteins (e.g., v-SNARE), and erroneously captured endoplasmic reticulum proteins from the cis-Golgi network back to the rER. Transport vesicles that fuse with the plasma membrane and release their contents into the intercellular space are called secretory or exocytic vesicles. Along with coated vesicles, coated pits also form within the cell, i.e., endocytic vesicles that bud inward from coated Regions of the plasma membrane.

When a cell needs to "fish out" a substance from the extracellular fluid, it typically employs The Mechanism of receptor-mediated endocytosis, or adsorptive endocytosis, in which the cell expresses receptor proteins for this substance that subsequently incorporate into the coated regions of the plasma membrane. All molecules bound to the receptors rapidly enter the cell because coated pits constantly pinch off inward to form coated vesicles.

These vesicles rapidly lose their coat and fuse with other vesicles to form larger vesicles called endosomes. The contents of endosomes can be utilized by the cell. A well-studied example of adsorptive endocytosis is the uptake of Cholesterol from the extracellular environment by animal cells.

Some transport vesicles that bud off from the outermost cisterna of the trans-side of the Golgi apparatus become primary lysosomes. The glycoproteins loaded into these vesicles feature an oligosaccharide with a phosphorylated mannose residue (Figure 87).

Figure 87 - Mannose-6-phosphate

The mannose-6-phosphate marker determines the targeting of these glycoproteins to lysosomes. Following the Cleavage of the mannose-6-phosphate marker, the glycoproteins of primary lysosomes become active hydrolytic enzymes (i.e., cleavage enzymes that transfer functional groups to a water molecule).

Lysosomes break down macromolecules captured by the cell via endocytosis into monomeric components, as well as distinct regions of the Cytoplasm and Organelles that have lost their significance As a result of Aging or are utilized as material to maintain cell viability under adverse extreme conditions.

In the first case, the primary lysosome fuses with an endocytic vesicle. In the second case, the portion of the cytoplasm or organelle destined for "Digestion" is first surrounded by a membrane, after which the resulting vesicle fuses with the primary lysosome.

A primary lysosome that has fused with a vesicle containing a substrate for breakdown is referred to as a secondary lysosome. The monomeric degradation products are transported from the lysosome into the cytoplasm and can be utilized by the cell.

Over 60 hydrolytic enzymes are currently known to be contained in lysosomes, capable of breaking down virtually all natural polymeric Organic compounds. All of them exhibit maximum activity at pH ≈ 5. It is precisely this pH level that is maintained inside the lysosome by V-type ATPases (proton pumps) integrated into their membrane, which utilize ATP energy to pump H⁺ ions into the lumen of these organelles. Although under normal conditions the lysosomal membrane is impermeable to hydrolytic enzymes, the requirement for an acidic environment for their active function protects the Cell Cytoplasm from destruction in the event of a potential enzyme "leakage".

METABOLISM/35.html">Selection/41.html">Review Questions and tasks

1. Characterize the Three Main Mechanisms of intracellular protein transport.

2. What are sorting signals?

3. Which sequences are referred to as targeting sequences?

4. WHAT IS A signal peptide, and what cellular membrane process does a signal peptide initiate?

5. Which cellular process is blocked if a KDEL sequence is present at the C-terminus of a protein?

6. What components make up the nuclear pore complex?

7. What states can a GTPase exist in?

8. Which auxiliary proteins regulate GTPase activity?

9. Describe the stages of Ran-GTPase-mediated protein import into the nucleus through the nuclear pore.

10. Describe the stages of Ran-GTPase-mediated protein export from the nucleus through the nuclear pore.

11. Describe the features of Protein transport into Mitochondria.

12. Describe the Stages of Protein export into Peroxisomes.

13. Describe the Stages of Protein Synthesis in the rough endoplasmic reticulum.

14. What function do coat proteins perform in vesicular transport?

15. List three types of coat proteins.

16. What is the function of SNARE Protein Complexes?

17. To which organelles are glycoproteins bearing a mannose-6-phosphate tag targeted?