(a) What is rough endoplasmic reticulum (RER)? Describe in detail the various modifications of secretory proteins occurring in…

(a) Rough endoplasmic reticulum and modification of secretory proteins in its lumen

The rough endoplasmic reticulum (RER) is the part of the ER made of flattened cisternae whose cytosolic face is studded with ribosomes. It is continuous with the outer nuclear membrane and makes secretory, lysosomal and membrane proteins.

Entry into the lumen: the N-terminal signal sequence of a nascent secretory protein is bound by the signal recognition particle, which takes the ribosome to the SRP receptor and the Sec61 translocon, and the chain is threaded into the lumen as it is made.

Modifications in the lumen:

1. Signal peptide cleavage: signal peptidase removes the signal sequence.
2. N-linked glycosylation: oligosaccharyltransferase transfers a preformed Glc₃Man₉GlcNAc₂ unit from dolichol to an asparagine in the sequence Asn-X-Ser/Thr; glucosidases then trim the glucose residues.
3. Folding with chaperones: BiP (an Hsp70) binds exposed hydrophobic stretches; calnexin and calreticulin bind the monoglucosylated glycan and hold the protein until it is folded, and UGGT adds a glucose back to a misfolded one.
4. Disulphide bond formation: protein disulphide isomerase forms and rearranges S–S bonds, with Ero1 maintaining the oxidising environment.
5. Proline isomerisation: peptidyl-prolyl isomerase interconverts cis and trans prolines.
6. Other changes: hydroxylation of proline and lysine in procollagen (needs vitamin C) and assembly of subunits, for example the heavy and light chains of antibodies.

Quality control: correctly folded proteins leave in COPII vesicles for the Golgi. Misfolded ones, marked by mannose trimming (ER mannosidase), are retrotranslocated to the cytosol, ubiquitinated and degraded by the proteasome (ER-associated degradation); their accumulation triggers the unfolded protein response. Resident luminal proteins such as BiP and protein disulphide isomerase carry a C-terminal KDEL signal and are retrieved from the Golgi by the KDEL receptor.

(b) Endomitosis and the polytene chromosome

Endomitosis is repeated duplication of the chromosomes within an intact nuclear envelope, without spindle formation, nuclear division or cytokinesis, so the cell becomes polyploid. Polytene chromosomes arise by the closely related endoreduplication, in which repeated S phases occur and the daughter chromatids stay together.

Structure (larval salivary glands of Drosophila and Chironomus):

• Each chromosome is a giant cable of up to 1,024 (2¹⁰) chromatids lying side by side in register after ten rounds of replication, about 100 times thicker than an ordinary chromosome and visible under the light microscope.
• Homologues are paired (somatic synapsis), so only the haploid number of chromosomes is seen (four in Drosophila: X, 2, 3 and 4), with their arms joined at a chromocenter formed by the fused centromeric heterochromatin.
• A constant pattern of dark bands (tightly coiled chromomeres rich in DNA) alternates with light interbands (uncoiled DNA); Drosophila has about 5,000 bands. The pattern is used for cytological maps and to locate deletions, duplications and inversions.

Puffs are swellings at some bands, where the chromatids uncoil into loops; the very large puffs of Chironomus are Balbiani rings. Their importance:

• they are sites of active transcription, rich in RNA and RNA polymerase II, and take up labelled uridine;
• the puffing pattern differs between tissues and stages, so it shows differential gene activity;
• ecdysone induces puffs in a fixed sequence (early, then late puffs), and heat shock induces puffs at heat-shock genes, so puffs reveal gene regulation by hormones and stress.

(c) Facilitated diffusion and active transport across the plasma membrane

Facilitated diffusion is the passive movement of polar or charged solutes (glucose, amino acids, ions) down their concentration or electrochemical gradient through specific channel or carrier proteins. It needs no metabolic energy, is specific and saturable, and can be blocked by inhibitors. Example: entry of glucose through the uniporter GLUT1, a carrier that alternately opens to either side of the membrane.

Active transport moves a solute against its gradient and needs energy. In primary active transport the pump itself hydrolyses ATP. Example: the Na⁺–K⁺ ATPase (a P-type pump of α and β subunits), which works in a cycle:

1. In the inward-facing form with ATP bound, it binds 3 Na⁺ from the cytoplasm.
2. ATP phosphorylates a conserved aspartate of the pump; the protein changes conformation, opens to the outside and releases the 3 Na⁺.
3. The outward-facing pump binds 2 K⁺ from outside, and the phosphate is removed.
4. The protein returns to the inward-facing form and releases the 2 K⁺ into the cytoplasm.

Each ATP thus moves 3 Na⁺ out and 2 K⁺ in. The pump is inhibited by ouabain; it maintains the Na⁺ and K⁺ gradients and the membrane potential, and controls cell volume. Other pumps include the Ca²⁺ ATPase of the sarcoplasmic reticulum and the H⁺–K⁺ ATPase of the gastric mucosa.

In secondary active transport the downhill Na⁺ gradient drives the uphill movement of another solute; for example, the Na⁺–glucose symporter SGLT1 of the intestinal epithelium takes up glucose together with two Na⁺. Unlike facilitated diffusion, which stops at equilibrium, active transport can build up a gradient.