Membranes and Membrane Transport Flashcards

Question

Nuclear transport occurs through. . .

Answer 1

Nuclear pores complexes Attached is an image showing scanning EM of both sides of a nuclear pore. Note the 8-fold symmetry.

Answer 2

The nuclear localization sequence is a binding site for **importins**. The nuclear pore proteins **(nucleoporins) have long tails** that have repeats containing hydrophobic amino acids like **phenylalanine**. These hydrophobic tails stick together due to the hydrophobic effect, creating a **meshwork that blocks the passage** of moderate- to large-sized proteins. The **importin** proteins have **hydrophobic amino acids on their surface** that can interact with the hydrophobic tails of the nucleoporins, **allowing them to “melt”** the meshwork **and pass through it**. By binding cargo proteins with NLS’s, the importins can piggyback proteins through the meshwork.

Answer 3

SKL = Serine-Lysine-Leucine

Answer 4

All of these proteins are considered peripheral membrane proteins, but only the **proteins on the inside** of the cell would be **translated by cytosolic ribosomes**. The **protein on the outside** of the cell would be **translated by a ribosome attached to the ER.**

Answer 5

Channel in the ER membrane that binds proteins by the ER localization sequence as they are being translated and facilitates their translation **into** the ER.

Answer 6

The signal sequence is cleaved off after successful import in the ER.

Answer 7

**Disulfide bonds can only form in the ER** and not the cytosol which has a high concentration of reducing agents. Disulfide bridge formation is catalyzed by protein-disulfide isomerase (PDI). It itself becomes reduced in the process and therefore needs to be **re-oxidized.** The responsible oxidase is **Ero1 (ER oxidase 1)**, which uses **molecular oxygen.** If an **erroneous disulfide bridge** is formed, the protein will get stuck in a high-energy state, most likely partially unfolded, **so oxidized PDI may reach and reduce the disulfide bond**.

Answer 8

When the protein reaches the Golgi, some additional mannose sugars are removed, and other sugars are added. This is referred to as “**peripheral glycoslylation**”. NANA=N-acetylneuraminic acid (a sialic acid) Gal=Galactose (a sugar) Man=mannose (a sugar) GlcNAc=N-acetylglucosamine (a sugar) Fucose (sugar) Note that is therefore possible to determine where a protein is located in the secretory pathway by analyzing the pattern of carboyhydrates attached to the protein.

Answer 9

Direct a protein in the Golgi away from the constitutive secretion pathway and towards a regulated secretion pathway.

Answer 10

1. Budding 2. Targeting 3. Fusion

Answer 11

1. Alter the shape of the membrane for pinching off 2. Bind the cargo to be transported 3. Make release an irreversible process so fusion may occur

Answer 12

The first step is that a protein called **Sar1** is recruited to the cytoplasmic face of the ER. Sar1 is a **GTP-binding protein.** When Sar1 binds to GTP, it **exposes** a short amphipathic **helix** that interacts with the membrane. This causes **Sar1 to bind to the membrane** (yellow protein). Sar1 in turn **interacts with other proteins** (red), causing them to be localized together into a patch on the ER membrane. These proteins in turn interact with proteins that span the membrane (green). These are the **cargo proteins** that will be recruited into the vesicle. In addition, **Sar1** binds to proteins that form the **outer coat** (orange). These proteins help bend the membrane to **pinch off** the vesicle. The lumen of the vesicle also contains other cargo proteins that bind to the transmembrane proteins (green). To **release upon fusion**, Sar1 can **hydrolyze the GTP** that it binds, and when it does so, it changes shape. This causes the whole **coat protein complex to fall apart,** leading to a naked vesicle that can fuse with the target membrane.

Answer 13

**Fusion is mediated by the SNAREs**. Each vesicle and each acceptor compartment contain a SNARE protein (called either **v-** or **t-SNARE** for **vesicle** and **target**) that pair with each other. Formation of the v-, t-SNARE complex is believed to drive fusion. Notice that the t-SNARE is composed of 3 separate polypeptide chains. The SNAREs all have one domain embedded in the membrane and one domain in the cytosol. The cytosolic domains interact with one another to promote fusion.

Answer 14

An enzyme uses **chemical energy from ATP** to **separate the proteins** (since the enzyme hydrolyzes ATP in the process it is generically referred to as an ATPase). Once the proteins are separated (lower panel), the V-SNAREs have to be returned to their appropriate compartment so that they can be used again. The T-SNARES can remain where they are, since they are already in the target membrane (by definition)

Answer 15

Clathrin forms coated pits - membrane indentations that are covered on the cytoplasmic side by clathrin. Clathrin can form regular lattice structures that can assemble to form cages. It is thought that the **formation of these cages is what drives vesicles to bud from the plasma membrane**. After budding the clathrin coat is **quickly disassmebled** with the help of **Hsp70**, an **ATP driven molecular chaperone that resides in the cytosol.**

Answer 16

For proteins that are trafficked to the lysosome, they are **modified differently in the Golgi apparatus**. The figure on the left shows the carbohydrate modification of the protein as it arrives from the ER. In the Golgi, and enzyme called **GlcNac phosphotransferase** uses a molecule of U**DP-N acetyl glucosamine** to **transfer a phosphate** to one of the mannose side chains (at position 6 of the mannose, so it is called **mannose-6 phosphate**). The mannose 6-phosphate is the key tag that helps the protein get sorted to the lysosome. This means that the enzyme GlcNac phosphotransferase has to know what proteins are bound for the lysosome and which are not. The enzyme only binds to those proteins that are destined to go to the lysosome. **Instead of a single short sequence** (like an ER signal sequence), the information encoding whether a protein is destined to the lysosome is more complex, and is conveyed in the **overall 3D shape of the protein**. Those proteins that have the right sequences and shapes to bind to GlcNac phosphotransferase get modified and thus targeted to the lysosome. This method of targeting represents a slightly different concept in trafficking: instead of recognizing only a short sequence motif, this mechanism recognizes a folded shape. **Note that this method therefore also requires that the protein be folded properly before being modified and sorted to the lysosome.**

Answer 17

In the trans-Golgi network, proteins that bear the M6P sorting signal interact with **M6P receptors** in the membrane and thereby are **directed into clathrin/AP1 vesicles** (step 1). The coat surrounding released vesicles is rapidly **depolymerized** (step 2), and the uncoated transport vesicles **fuse with late endosomes** (step 3). After the phosphorylated enzymes dissociate from the M6P receptors and are **dephosphorylated**, late endosomes subsequently **fuse with a lysosome** (step 4). Note that coat proteins and M6P receptors are **recycled** (2 and 4a), and some receptors are delivered to the **cell surface** (step 5). Phosphorylated lysosomal enzymes occasionally are **sorted from the trans-Golgi to the cell surface and secreted**. These secreted enzymes can be retrieved by receptor-mediated endocytosis (steps 6-8), a process that closely parallels trafficking of lysosomal enzymes from the trans-Golgi network to lysosomes.

Answer 18

Assembles (+ GTP) and disassembles (GTP hydrolysis) coat proteins

Answer 19

Untangles SNAREs