Protein Localization and Secretion Notes

Protein Localization

• Proteins made in the cytoplasm need to be targeted to the correct location within the cell.

• There are three main pathways for proteins synthesized in the cytoplasm to reach their destinations:

  • Through nuclear pore complexes (NPCs) into the nucleus.

  • Across membranes into specific target organelles.

  • Entry into the endoplasmic reticulum (ER), followed by packaging into vesicles for transport.

Signal Sequences

• Signal sequences are like addresses that specify where a protein needs to be delivered within the cell.

• Examples of signal sequences include:

  • Import into ER: H_2N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-

  • Retention in the lumen of the ER: Cys-Glu-Val-Phe-Gln--Lys-Asp-Glu-Leu-COO-

  • Import into mitochondria: H_2N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-

  • Import into the nucleus: -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-

  • Import into peroxisomes: -Ser-Lys-Leu-

• Positively charged amino acids are often shown in red, negatively charged in blue, and hydrophobic amino acids in green.

• H_2N: N-terminus of a protein; COO-: C-terminus.

• The ER retention signal, KDEL, is a common example.

Nuclear Envelope and Nuclear Pore Complex (NPC)

• The nuclear envelope comprises a double membrane system: the inner and outer nuclear membranes.

• The outer nuclear envelope is continuous with the ER.

• The NPC facilitates transport into and out of the nucleus.

• NPC Structure:

  • Contains ring proteins, cytosolic fibrils, scaffold nucleoporins, channel nucleoporins, and a nuclear basket.

  • The NPC spans both the inner and outer nuclear membranes.

  • Includes cytosolic fibrils, a ring subunit, and nuclear fibrils and basket.

Nuclear Import

• GTP hydrolysis by the small GTPase Ran provides the energy for nuclear import and export.

• Molecules smaller than 50 kDa can diffuse through the NPC.

• Energy is required to enrich smaller molecules or move larger molecules through the NPC.

Nuclear Localization Signal (NLS)

• A short amino acid sequence can specify nuclear location.

• Experiment demonstrating necessary and sufficient components for nuclear localization.

Demonstrating Necessary and Sufficient NLS

• Experiments were conducted to determine what is necessary for nuclear localization:

  • Duplicated wild-type (WT) sequence goes to the nucleus.

  • Lysine at position 128 (K128) is necessary.

  • The presence of threonine at position 128 (T128) does not retain the sequence in the cytosol.

  • Putting NLS at the N-terminus still requires K128.

Defining Minimal NLS Sequence with Pyruvate Kinase

• Experiments using pyruvate kinase (PK) to define the minimal NLS sequence:

  • Sequences of various XR-PK constructs and their localization patterns (N = nucleus, C = cytoplasm).

  • XR40-PK: MET ASP LYS VAL PHE ARG ASN SER SER ARG THR PRO PRO LYS LYS LYS ARG LYS VAL GLU ASP PRO ARG ASN SER

  • Additional constructs with varying sequences and localization.

Endoplasmic Reticulum (ER)

• The ER is a dynamic organelle. ER can be rough (containing ribosomes) or smooth.

Biochemical Isolation of ER

• Rough ER (ER with Ribosomes) + Salt -> Centrifuge results in Ribosomes + mRNA and ER + mRNA + Protein (Ribosome) No Protein, but Protein present with Rough ER.

• Microsomes: Low density with low sucrose concentration and high density with high sucrose concentration.

Further Evidence of the Signal Hypothesis

• Rough ER (ER+Ribosomes) +Salt Centrifuge Ribosomes +mRNA ER +mRNA +mRNA Protein (Ribosome) No Protein

• Protein (Rough ER) SDS-PAGE Ribosome Protein

• Rough ER ProteinResult: Why?

Differences from NLS

• Cytosolic Protein: mRNA remains free in cytosol.

• ER-targeted Protein: mRNA encoding a protein targeted to ER remains membrane-bound. Target protein is co-translationally transported into the ER lumen.

• Import into ER: H_2N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln-

Signal Recognition Particle (SRP)

• The Signal Recognition Particle (SRP) binds to signal sequence slows translation and brings the ribosome to the ER via binding to the SRP receptor.

• Translational Pause Domain: part of the SRP RNA molecule.

• Hinge region of SRP.

Sec61 Complex

• The Sec61 complex forms the core of the ER channel.

• Functions:

  • Allows proteins to pass through an aqueous channel once engaged with the ribosome.

  • Opens a hinge to allow signal sequences to diffuse into the ER lipid bilayer.

Protein Depositioning into the ER Lumen

• Soluble proteins deposited into the ER lumen, requires cleavage of signal peptide and KDEL sequence for retention in ER Lumen.

• Transmembrane proteins require hydrophobic "START" and "STOP" sequences.

• Multipass transmembrane proteins require internal "START" followed by a "STOP".

ER Orientation

• Keep orientation straight as things move throughout the cell. Inner versus outer leaflets? Secreted versus cytosolic? N versus C-terminal in the cytosol versus lumen/extracellular?

Vesicular transport

• COP-Coated Vesicles, Clathrin-coated Vesicles from Golgi.

• Budding then Fusion.

Secretory Mutants in Yeast

• Genetic screen in budding yeast identified temperature-sensitive mutants affecting secretion.

• Secreted protein versus internal protein were examined using Acid Phosphatase and Invertase.

• Cyclohexamide: blocks translation.

SEC1 Mutant

• Sec1 mutants stop growing at restrictive temperature but continue to make protein.

• Sec1 mutants accumulate vesicles -> protein that should be secreted is in the vesicles.

SEC Genes

• SEC1 encodes a SNARE binding protein.

• Genetic studies identified numerous SEC genes involved in different stages of the secretory pathway.

• Functions of other SEC genes include:

  • Vesicle budding from ER and Golgi.

  • Tethering of vesicles to target membranes.

  • Fusion of vesicles with target membranes.

  • Recycling of SNAREs.

SNAREs

• SNAREs (Soluble NSF Attachment protein REceptors) mediate vesicle fusion.

• v-SNARE = Vesicle SNARE, t-SNARE = Target Membrane SNARE

• Tight interactions between SNAREs bring vesicle and target membrane very close together (<1.5 nm). Lipid bilayers can then fuse together.

Neurotransmission

• Electrical signal of an action potential converted to chemical signal via voltage-gated Ca^{2+} channels.

• Chemical signal converted back to an electrical signal via ligand/neurotransmitter-gated ion channels.

SNAREs in Synaptic Vesicle Fusion

• Fusion of vesicles in the nerve termini of the pre-synaptic cell is mediated by SNAREs.

• Calcium-binding protein triggers fusion of the synaptic vesicle with the plasma membrane.

• Upon fusion, contents of synaptic vesicle released into the synaptic cleft.

Motors and Transport

• Motors and transport mechanisms facilitate the movement of proteins and vesicles within the cell.