Fundamentals of Molecular Biology. Part 1: Molecular Biology of the Cell - A.N. Ogurtsov 2011

Intracellular Transport of Substances
Vacuolar 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 endoplasmic reticulum, while the vesicles themselves contain Soluble Glycoproteins captured from the lumen of the endoplasmic reticulum.

Most of these vesicles fuse to form flattened cisternae on the side of the Golgi apparatus closest to The Nucleus, known as the forming or cis-face (forming pole).

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

Protein "maturation" involves various covalent modifications through which proteins acquire their functionally active Structure. In addition, the Oligosaccharides linked to asparagine residues, which were previously attached to the proteins in the endoplasmic reticulum, undergo extensive modification: some sugar residues are selectively cleaved, 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 responsible for protein modification, much like the proteins being modified, likely enter the Golgi apparatus from the endoplasmic reticulum. It is currently believed that following processing, the oligosaccharide attached to the protein serves as an "address" determining the protein's final destination.

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

A GENERALIZED SCHEME OF Vesicular Transport is presented in Figure 34. Proteins synthesized in the rough endoplasmic reticulum (Figure 34(1)) are packaged into transport vesicles (Figure 34(2)), which bud off from the rough endoplasmic reticulum and fuse together to form a new cis-Golgi network (CGN) cis-cisterna. Enzymes and Proteins of the endoplasmic reticulum that entered such vesicles, as well as the membrane proteins responsible for vesicle formation, are returned to the rough ER for "reuse" (Figure 34(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 34 — Generalized scheme of vesicular transport

Each Golgi cisterna from the cis-region physically moves through the medial region into the trans-region of the Golgi apparatus via a non-vesicular process known as cisternal progression (Figure 34(4)).

The "retrieval" of proteins specific to each region of the Golgi apparatus is accomplished by a vesicular mechanism (Figure 34(5)). In all Cells, certain proteins are continuously transported to The Cell surface in vesicles budding from the trans-face of the Golgi apparatus, ensuring constitutive protein secretion (Figure 34(6)) outside the cell (constitutive exocytosis). In certain cell types, specific proteins are "stored" in secretory vesicles (Figure 34(7)) and are secreted only upon receiving an external neural or hormonal signal (regulated exocytosis).

Proteins and lipids destined for Lysosomes (Figure 34(8)) bud off from the trans-face of the Golgi apparatus and join secondary endosomes, which subsequently fuse with a lysosome. Proteins from the extracellular space and Plasma Membrane proteins that form endocytic vesicles during endocytosis (Figure 34(9)) (pinching off from The Plasma Membrane into the interior of the cell) are also transported into lysosomes via endosomes.

Coated vesicles. Most transport vesicles represent a special class of intracellular vesicles that appear under micrographs 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) COP-protein coats vesicles transporting proteins from the rough endoplasmic reticulum to the Golgi apparatus,

2) COPI-protein coats vesicles mediating the "retrograde" transport of proteins between Golgi cisternae and back to the rough endoplasmic reticulum,

3) clathrin coats vesicles transporting proteins from the plasma membrane and the trans-Golgi network to secondary endosomes (Figure 35).

Figure 35 — STRUCTURE OF THE clathrin coat: a — triskelion; b — packing of triskelions into the clathrin coat; c — clathrin cages formed by spontaneous aggregation of triskelions in vitro; 1 — clathrin; 2 — small polypeptide; 3 — clathrin-binding site during aggregation

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

The primary structural element of the coat is a protein complex called a triskelion, consisting of three clathrin polypeptide chains and three small (35 kDa) Polypeptides.

On The surface of coated vesicles, triskelions form basket-like networks of hexagons and pentagons. Isolated triskelions are capable of spontaneous aggregation under appropriate conditions. Even in the absence of vesicles, this process forms typical polyhedral cages (Figure 35(b)).

Numerous accessory proteins are integrated into the vesicle membranes. These proteins, firstly, "capture" proteins with matching oligosaccharide "addresses" from the Golgi cisternae during vesicle formation, and secondly, recognize the Components of the target membrane with which the vesicle must fuse (Figure 36).

It is known that the contents of each transport vesicle are delivered precisely to their correct "address" at a specific intracellular membrane. Therefore, there must be distinct subpopulations of transport vesicles carrying unique accessory proteins on their surface—so-called docking markers (v-SNARE proteins) (Figures 36 and 37)—which are recognized by complementary receptors (t-SNARE proteins) on target membranes to form the SNARE complex.

Figure 36 - 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 - SNARE complex

The coat plays a crucial role in vesicle formation, but shortly after the vesicle buds off, the coat proteins dissociate from its surface (uncoating). 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 network is shown in Figure 37.

Direct (anterograde) transport is mediated by COPII-coated vesicles (Figure 37(1, 2, 3)), which are formed by the polymerization of COPII proteins on the surface of the endoplasmic reticulum membrane.

Figure 37 - Vesicular protein transport between the endoplasmic reticulum and the cis-Golgi region

The dissociation of COPII proteins from the vesicle surface exposes the 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.

Reverse (retrograde) transport (Figure 37(4, 5, 6)), carried out by COPI-coated vesicles, ensures the return (recycling) of Membrane Lipids, specific proteins (e.g., v-SNARE), and mistakenly 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 extracellular space are called secretory or exocytic vesicles. Alongside coated vesicles, cells also form coated pits—that is, endocytic vesicles that bud inward into the cell from coated Regions of the plasma membrane.

When a cell needs to "capture" a specific substance from the extracellular fluid, it typically utilizes receptor-mediated endocytosis, or adsorptive endocytosis. In this process, the cell produces receptor proteins for that substance, which become integrated into the coated areas of the plasma membrane. All molecules bound to the receptors rapidly enter the cell because the coated pits continuously pinch inward to form coated vesicles.

These vesicles rapidly shed their coats and fuse with other vesicles to form larger structures 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.

Certain transport vesicles budding from the outermost cisternae of the trans-Golgi network become primary lysosomes. The glycoproteins loaded into these vesicles feature an oligosaccharide with a phosphorylated mannose residue (Figure 38). This mannose-6-phosphate tag determines The Fate of such vesicles. Following the Cleavage of the mannose-6-phosphate marker, the primary lysosomal glycoproteins become active hydrolytic enzymes (i.e., digestive enzymes that transfer functional groups to water molecules).

Figure 38 - Mannose-6-phosphate

Lysosomes break down macromolecules captured by the cell via endocytosis into monomeric components, as well as specific regions of the Cytoplasm and Organelles that have become obsolete due to Aging or are used as material to sustain cell viability under unfavorable, extreme conditions.

In the first case, a primary lysosome fuses with an endocytic vesicle. In the second case, the portion of cytoplasm or organelle destined for "Digestion" is first enclosed 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 products of degradation are transported from the lysosome into the cytoplasm and can be utilized by the cell.

Approximately 60 hydrolytic enzymes are currently known to be housed within lysosomes, capable of breaking down virtually all natural polymeric Organic compounds. All of them exhibit peak activity at pH ≈ 5. This acidic pH is maintained inside the lysosome by proton ATPases (proton pumps) embedded in 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 acidic environment required for their optimal function protects the Cell Cytoplasm from destruction in the event of potential enzyme "leakage."