Protein Targeting

Table of Contents
1. Protein Targeting and Intracellular Transport
2. Co-Translational Targeting
3. Protein Sorting to the ER
4. Translocation of Secretory Proteins Across the ER Membrane
5. Insertion of Proteins into the ER Membrane
6. Protein Modifications, Folding and Quality Control in the ER
7. Golgi Apparatus
8. Vesicle Transport
9. Lysosome Targeting
10. Post-Translational Transport
11. Peroxisome Targeting
12. Key Points on Peroxisomes
13. Nucleus
14. Perspective for the Future
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Protein Targeting and Intracellular Transport

• Protein targeting directs proteins that must function outside the cytosol to their correct cellular location.
• Targeting is more complex in eukaryotes because they contain many internal compartments.
• Decisions about a protein's destination are made while the protein is being synthesised.
• The information for targeting is encoded in the nascent protein sequence and may be removed later by proteolytic processing.
• Membrane proteins and water-soluble proteins are sorted differently: membrane proteins are inserted into lipid bilayers, while soluble proteins are translocated across membranes into organelle lumens.
• In eukaryotic cells, major destinations include nucleus, mitochondria, peroxisomes, chloroplasts, endoplasmic reticulum (ER), Golgi apparatus, lysosomes and secretory vesicles.
• Regions on proteins that mediate targeting are called sorting signals or signal sequences.
• Sorting signals are specific amino-acid stretches that bind receptors on target organelles or carrier proteins.

There are two basic classes of targeting pathways.

1. Co-translational targeting (secretory pathway) - routes proteins into ER, Golgi, lysosome, plasma membrane and secreted proteins.
2. Post-translational targeting - routes proteins to nucleus, mitochondria, chloroplast and peroxisome.

Co-Translational Targeting

Endomembrane System Targeting

• The endomembrane system (EMS) is a three-dimensional network of vesicular compartments that mediate trafficking in eukaryotic cells.
• The EMS includes the ER, Golgi apparatus, endosomes and lysosomes and is sometimes referred to as the GERL complex.
• Organelles that do not form from the ER are not considered part of the EMS.

Endoplasmic Reticulum (ER)

• The ER is an extensive interconnected network of flattened cisternae and tubules bounded by membrane.
• ER membranes are continuous with the outer nuclear membrane and connect with the plasma membrane at other sites.
• ER is present in almost all eukaryotic cells except mature red blood cells and sperm.
• The ER functions as a site for synthesis, folding and transport of many proteins and works closely with ribosomes, mRNA, tRNA and the Golgi apparatus.
• ER size and organisation vary with cell function; secretory cells such as liver and pancreatic cells have abundant ER.
• Prokaryotes and certain differentiated cells lack ER.

ER Structural Elements

• Cisternae: long, flat sac-like membrane plates arranged in parallel; the enclosed cisternal lumen is continuous with the perinuclear space but separate from the cytosol.
• Vesicles: round or ovoid sacs that transport ER cargo to the Golgi complex.
• Tubules: irregular branched tubular elements, 50-100 nm diameter, surrounded by a unit membrane and filled with secretory products.

Types of ER

• Smooth endoplasmic reticulum (SER): lacks ribosomes and participates in steroid and lipid synthesis, glycogen metabolism, carbohydrate metabolism, detoxification and intracellular calcium storage and release.
• Rough endoplasmic reticulum (RER): ribosome-studded surface; active in protein synthesis, translocation, folding, glycosylation, disulfide-bond formation and membrane synthesis.

Common ER Functions

• Provide structural framework and increased surface area for cellular reactions.
• Participate in active transport of cellular materials and metabolic activities via resident enzymes.
• Contribute membranes for nuclear envelope formation during cell division.
• Microsomes are vesicles derived from fragmented ER; rough microsomes retain ribosomes and smooth microsomes do not.

Protein Sorting to the ER

• Proteins that enter the secretory pathway are initially targeted to the ER membrane and include soluble luminal proteins, ER-resident membrane proteins, secreted proteins and many proteins destined for Golgi, lysosomes or the plasma membrane.
• Transport along the secretory pathway occurs in small vesicles that bud from one organelle and fuse with the next compartment.
• The signal-sequence hypothesis, first proposed by Günter Blobel, explains how signal sequences direct proteins into the ER.
• Each organelle has receptor proteins that recognise specific signal or uptake sequences to ensure targeting specificity.
• After receptor binding, a polypeptide is transferred to a translocation channel that allows unidirectional passage across the membrane.
• Translocation often requires energy, for example from ATP hydrolysis, and signal sequences are frequently removed by proteases after translocation.

Translocation of Secretory Proteins Across the ER Membrane

• Protein synthesis begins on free cytosolic ribosomes for all proteins.
• Ribosomes synthesising secretory or membrane proteins are targeted to the ER by an N-terminal signal sequence on the nascent polypeptide.
• Signal sequences are short hydrophobic stretches (about 6-12 residues) that are usually cleaved by signal peptidase in the ER lumen.
• Most secretory proteins enter the ER lumen while still being synthesised on the ribosome, a process called co-translational translocation.

Signal Hypothesis

• As the nascent chain emerges from the ribosome, the signal sequence is recognised by the Signal Recognition Particle (SRP), a cytosolic ribonucleoprotein.
• SRP comprises six polypeptides and an approximately 300-nucleotide RNA and binds both the ribosome and the signal sequence.
• SRP binding pauses translation and targets the ribosome-nascent chain complex to the ER by binding the SRP receptor on the ER membrane.
• SRP proteins P9 and P14 interact with the ribosome, while P68 and P72 contribute to translocation.
• SRP blocks the elongation factor binding site on the ribosome and thereby halts translation until docking at the translocon.
• The translocon (Sec61 complex in eukaryotes, composed of α, β and γ subunits) accepts the nascent chain and inserts the signal sequence into a protein-conducting channel.
• GTP binding and hydrolysis on SRP and the SRP receptor coordinate transfer; GTP hydrolysis causes SRP to dissociate and translation to resume, allowing the polypeptide to enter the ER lumen.
• In yeast some secretory proteins can be translocated post-translationally without SRP; such cases use a Sec63 complex and the lumenal chaperone BiP.
• BiP is an Hsp70-family chaperone containing peptide-binding and ATPase domains that bind and stabilise unfolded proteins during translocation.

Insertion of Proteins into the ER Membrane

• Membrane proteins adopt a specific orientation that is preserved during and after insertion: the same segment faces the cytosol and other segments face the lumen.
• Topogenic sequences determine insertion and orientation of integral membrane proteins.
• Single-pass proteins (topology classes I, II and III) have one transmembrane α-helix, while multi-pass proteins (class IV) contain several transmembrane segments.
• Type I single-pass proteins typically have an N-terminal signal sequence followed by a luminal domain and then a transmembrane hydrophobic segment that anchors in the membrane.
• Type II single-pass proteins often have an internal signal-anchor sequence that results in the N-terminus remaining cytosolic and the C-terminus luminal.
• Hydropathy plots help identify hydrophobic segments that act as signal sequences or transmembrane anchors.

Hydropathy Plot of Connexin

• A hydropathy index plots the relative hydrophobicity of successive segments of a polypeptide chain.
• Positive peaks indicate hydrophobic regions and negative values indicate hydrophilic regions.
• For connexin, four prominent hydrophobic peaks correspond to four transmembrane α-helices.
• Some proteins are synthesised as type I proteins on the ER and later acquire a GPI anchor that transfers their luminal domain to the membrane surface.

Protein Modifications, Folding and Quality Control in the ER

• The ER is the site of protein folding, assembly of multisubunit proteins, disulfide-bond formation, initial N-linked glycosylation and addition of glycolipid anchors.

Disulfide Bond Formation

• Protein disulfide isomerase (PDI) in the ER lumen catalyses disulfide-bond formation and rearrangement.
• PDI has an active site with two closely spaced cysteines that cycle between reduced dithiol and oxidised disulfide forms.
• In forming disulfide bonds, a substrate thiolate attacks an oxidised PDI disulfide to form a PDI-substrate intermediate.
• A second substrate thiolate then resolves this intermediate to form an intramolecular disulfide in the substrate and release reduced PDI.
• Reduced PDI can also catalyse rearrangement of improperly formed disulfide bonds by thiol-disulfide exchange until the most stable conformation is achieved.

N-Linked Glycosylation

• N-linked glycosylation occurs co-translationally as polypeptides are translocated into the ER.
• More than 90% of cellular glycosylation events are N-linked.
• The core N-linked oligosaccharide contains 14 sugars: 2 N-acetylglucosamine (NAG), 3 glucose and 9 mannose residues.
• The complete 14-sugar oligosaccharide is transferred en bloc to specific Asn residues of the nascent chain.
• The oligosaccharide precursor is assembled on a dolichol lipid and is attached by a pyrophosphate linkage that provides activation energy for transfer.
• Oligosaccharyl transferase (OST) transfers the oligosaccharide from dolichol to the Asn residue and is associated with each protein translocator in the ER membrane.

Golgi Apparatus

• The Golgi apparatus, discovered by Camillo Golgi, is the manufacturing and shipping centre of eukaryotic cells.
• In mammalian cells a Golgi complex is usually located near the nucleus and centrosome.
• Golgi stacks are linked by tubular connections that depend on microtubules; disruption of microtubules fragments the Golgi into individual stacks.
• Organization differs between organisms: yeast have multiple scattered Golgi stacks and plant Golgi depend on actin rather than microtubules.
• A Golgi stack consists of flattened membrane sacs called cisternae, arranged polarised from cis (entry) to trans (exit) faces.
• The cis-Golgi network (CGN) receives vesicles from the ER and the trans-Golgi network (TGN) packages proteins into vesicles destined for lysosomes, secretory vesicles or the plasma membrane.
• The Golgi is a major site of carbohydrate processing and final protein sorting.
• Proteins arriving from the ER undergo ordered covalent modifications as they move through Golgi cisternae: trimming of N-linked oligosaccharides and addition of new sugar residues.
• High-mannose oligosaccharides may be trimmed without new additions, while complex oligosaccharides gain galactose, sialic acid and sometimes fucose.
• Sialic acid is generally absent in plants.

O-Linked Glycosylation

• O-linked glycosylation occurs in the Golgi and attaches sugars to serine, threonine or tyrosine residues.
• Sugar residues are typically added sequentially, often starting with N-acetylglucosamine (NAG) linked to Ser/Thr.
• Many cytoplasmic and nuclear proteins are modified by single O-linked NAG residues; this modification is reversible and can compete with phosphorylation at the same sites.
• For example, transcription regulator c-Myc contains Thr residues that may be either glycosylated or phosphorylated.
• Monoglycosylation of nuclear proteins can influence nuclear localisation and protein-protein interactions.

Conclusion on Glycosylation

• Glycan structures attached to proteins vary between species and tissues.
• Protein glycosylation is mainly a eukaryotic property and becomes more complex in higher organisms.
• Lower eukaryotes such as yeast attach simpler sugars, while mammals have highly branched oligosaccharides.
• N-linked glycosylation is generally more complex than O-linked glycosylation.
• Defects in genes involved in N-glycosylation cause developmental disorders, including intellectual and motor impairments.

Significance of Glycosylation

• Glycosylation increases solubility of nascent glycoproteins and helps prevent aggregation.
• Small intracellular monosaccharide modifications can participate in signalling.
• For secreted proteins, glycans protect against proteases and nonspecific interactions.
• Cell-surface glycans mediate cell-cell recognition by binding specific lectins on neighbouring cells.
• Oligosaccharide trimming intermediates assist folding and target misfolded proteins for degradation, aiding quality control.
• Many red blood cell surface antigens arise from glycosylation patterns.

Vesicle Transport

• Vesicles that carry secretory proteins between ER, Golgi and other compartments are often coated with cytosolic coat proteins.
• COPII coats mediate budding from the ER to the Golgi; after budding vesicles lose the COPII coat and fuse to form tubular clusters.
• v-SNARE and t-SNARE interactions promote membrane fusion of vesicles and target membranes.
• COPI coats mediate retrograde transport from Golgi back to ER.
• Retrieval signals for ER membrane proteins are often C-terminal sequences such as KKXX, which bind COPI via KDEL receptors.
• Soluble ER-resident proteins commonly bear a KDEL (Lys-Asp-Glu-Leu) retrieval signal.
• KDEL receptors bind KDEL-containing proteins with higher affinity at Golgi pH and release them at the neutral pH of the ER.
• V-type ATPases maintain a pH gradient from neutral ER to acidic Golgi, which regulates KDEL receptor binding.
• Clathrin-coated vesicles mediate budding from the plasma membrane and trans-Golgi; they participate in endocytosis and transport from the TGN to endosomes or lysosomes.
• Before fusion with a target membrane, vesicles shed their coat.
• Coat recruitment GTPases, a family of monomeric GTPases, control assembly of different coats.
• Arf proteins promote COPI and clathrin coat assembly at Golgi membranes.
• Sar1 mediates COPII assembly at the ER membrane.
• Rab proteins confer specificity to vesicle targeting by directing vesicles to specific membranes and recruiting Rab effector proteins.
• SNARE proteins on vesicle (v-SNARE) and target membranes (t-SNARE) mediate membrane fusion by forming specific trans-SNARE complexes.
• Animal cells have at least 35 SNAREs, each associated with particular organelles or pathways.
• SNAREs are well characterised in neurons where they mediate synaptic vesicle docking and fusion during neurotransmission.

Lysosome Targeting

Lysosome Structure and Function

• Lysosomes, discovered by Christian de Duve, are membrane-enclosed organelles filled with hydrolytic enzymes active at acidic pH (~5.0).
• They maintain acidity using H+-ATPase pumps that translocate protons into the lumen.
• Lysosomal inner membrane proteins are glycosylated to protect them from lumenal hydrolases.
• Lysosomes allow intracellular digestion and recycling of macromolecules; digested products cross the lysosomal membrane for reuse in the cytoplasm.
• Lysosomal enzymes are synthesised on RER, transported to the Golgi and modified there before being packaged into lysosomal vesicles.
• Primary lysosomes contain hydrolases but no ingested material; secondary lysosomes contain material being digested.
• Deficiency of specific lysosomal enzymes causes storage diseases such as Tay-Sachs and Pompe disease.

Autophagy (Autophagocytosis)

• Autophagy is a self-degradative process in which cytoplasmic components and organelles are delivered to lysosomes for breakdown.
• Cells form double-membrane autophagosomes that capture cytoplasmic material and fuse with lysosomes for degradation.
• Autophagy maintains cellular health by removing damaged or unused components and recycling their constituents.

Endocytosis and Phagocytosis

• Endocytosis internalises extracellular material into endosomes that fuse with lysosomes for digestion.
• Phagocytosis engulfs large particles such as bacteria into phagosomes, which then fuse with lysosomes for degradation.

Protein Targeting to Lysosomes

• Lysosomal proteins are modified differently from secreted or plasma-membrane proteins during Golgi processing.
• Mannose residues on N-linked oligosaccharides are phosphorylated to generate mannose-6-phosphate (M6P) tags while proteins are in the cis-Golgi network.
• The M6P tag is recognised by M6P receptors in the TGN, which sort these proteins into vesicles destined for endosomes and lysosomes.
• Recognition of lysosomal proteins for M6P addition depends on folded protein determinants called signal patches.

Post-Translational Transport

Mitochondria

• The term mitochondrion derives from Greek words meaning \"thread\" and \"granule\" and was coined by C. Benda.
• Mitochondria are double-membrane organelles present in nearly all eukaryotic cells and vary greatly in number per cell.
• The outer mitochondrial membrane (OMM) is relatively permeable due to porin proteins and allows passage of molecules up to ~10 kDa.
• The inner mitochondrial membrane (IMM) forms cristae and houses the electron-transport chain and ATP synthase for oxidative phosphorylation.
• The intermembrane space lies between the two membranes and the matrix is the space enclosed by the inner membrane.

Matrix

• The matrix contains enzymes of the tricarboxylic acid (TCA) cycle, mitochondrial ribosomes (70S type), mitochondrial DNA and various proteins and metabolites.
• Succinate dehydrogenase is the only TCA enzyme not free in the matrix; it is located on the inner membrane.
• Mitochondrial DNA is typically circular, maternally inherited in many organisms, and encodes a subset of mitochondrial proteins plus rRNAs and tRNAs.

Origin and Evolution

• Mitochondria are semi-autonomous organelles that divide by binary fission and show several bacterial-like features.
• They possess double membranes, circular DNA and reproduce by fission, supporting an endosymbiotic origin from bacteria.
• Margulis proposed the endosymbiotic theory whereby an ancestral host cell acquired aerobic bacteria that became mitochondria.
• Mitochondrial DNA replication and division respond to the cell's energetic needs, and inheritance is often maternal in higher eukaryotes.

Mitochondrial Targeting and Import

• Mitochondria and chloroplasts are double-membrane organelles with internal compartments and some autonomous genetic machinery.
• Many mitochondrial precursor proteins are synthesised in the cytosol and imported in an unfolded state, usually with N-terminal targeting sequences.
• Mitochondrial targeting sequences (MTS) are amphipathic helices of 20-50 residues enriched in Ser, Thr, Arg and Lys on one face and hydrophobic residues on the other.
• MTS lack negatively charged residues such as Asp and Glu.
• Import requires outer membrane receptors and translocons in both outer and inner membranes.
• Cytosolic chaperones such as Hsc70 help keep precursors unfolded and target them to the mitochondrion.
• Translocase of the outer membrane (TOM) complexes, including Tom20, Tom22 and the general import pore Tom40, recognise and translocate precursors across the OMM.
• Specific receptor proteins mediate import of different classes of mitochondrial proteins.

Protein Import into the Mitochondrial Matrix

• Matrix-destined precursors pass through an inner membrane translocon such as Tim23/17 at contact sites where outer and inner membranes are close.
• After entry into the matrix, the N-terminal MTS is cleaved by a matrix protease.
• Matrix Hsc70, anchored to the translocon by Tim44, binds incoming polypeptides and uses ATP hydrolysis to drive import.
• Final folding often requires chaperonins; without them assembly and folding may fail.
• Three energy sources drive mitochondrial import: ATP hydrolysis by cytosolic and matrix Hsc70 chaperones, and the proton-motive force across the inner membrane.
• The matrix Hsc70-Tim44 system may function as a molecular motor that pulls polypeptides into the matrix.
• Only respiring mitochondria that maintain a proton gradient can efficiently import many precursor proteins.

Multiple Signals and Pathways to Sub-Mitochondrial Compartments

• Several pathways deliver proteins from the cytosol to the inner membrane, intermembrane space or matrix.
• One pathway uses the Tim23/17 machinery and targets proteins such as cytochrome oxidase subunits.
• A second pathway inserts proteins that have both matrix-targeting sequences and internal hydrophobic segments via Oxa1 after matrix import.
• A third pathway imports multipass inner-membrane proteins that lack N-terminal MTS and contain internal targeting signals, for example ADP/ATP carrier proteins.

Chloroplast

• Chloroplasts are double-membrane semiautonomous organelles of plants and algae that carry out photosynthesis.
• They contain chlorophyll in thylakoid membranes and use light energy to produce ATP and NADPH.
• Chloroplasts also contribute to fatty-acid and some amino-acid synthesis and are believed to have arisen from an endosymbiotic cyanobacterium.
• Chloroplasts contain their own circular DNA (ctDNA or plastome) and divide by binary fission; they are inherited and distributed to daughter cells during division.
• Typical chloroplasts are lens-shaped, roughly 5-8 μm in diameter and 1-3 μm thick, though size varies by species and cell type.

Chloroplast Membranes and Stroma

• Chloroplasts have three membrane systems: outer envelope membrane, inner envelope membrane and the thylakoid system.
• The stroma is the protein-rich aqueous matrix where the Calvin cycle fixes CO2 to make sugars.
• The outer membrane is semi-porous and allows small molecules to diffuse, while the inner membrane contains TIC translocons for protein import.
• Many stromal proteins are encoded in the nucleus, synthesised in the cytosol and imported with N-terminal stromal import sequences (SIS).

Intermembrane Space and Peptidoglycan Wall

• A thin intermembrane space separates the outer and inner chloroplast membranes.
• Some algal chloroplasts (glaucophytes) retain a peptidoglycan wall between the membranes, resembling cyanobacterial ancestors; such plastids are called muroplasts.

Thylakoids and Grana

• Thylakoids are membrane sacks that contain chlorophyll and host the light reactions of photosynthesis.
• In many vascular plants thylakoids are stacked into grana connected by stromal thylakoids.
• Protein complexes in the thylakoid membrane, including photosystems I and II and electron carriers, drive H+ pumping into the thylakoid lumen and generate a proton gradient for ATP synthesis.
• The Calvin cycle enzymes reside in the stroma; the large subunit of Rubisco is encoded by chloroplast DNA, while the small subunit is nuclear-encoded and imported into the stroma.
• Stromal import involves N-terminal SIS sequences that are removed after import and chaperone-assisted folding by stromal Hsp70 and Hsp60 systems.
• Like mitochondria, chloroplast import depends on energy but does not use a proton-motive force across the inner membrane; import into the stroma appears to be powered mainly by ATP hydrolysis.
• Thylakoid-targeted proteins have secondary targeting sequences that are revealed after stromal import and use translocation pathways related to bacterial inner-membrane systems; one pathway can translocate folded proteins.

Peroxisome Targeting

• Peroxisomes are single-membrane organelles, typically 0.2-1.5 μm diameter, found in nearly all eukaryotic cells.
• They contain enzymes for lipid metabolism and detoxification and harbour both peroxide-producing and peroxide-destroying enzymes such as catalase.
• Peroxisomes originate from the ER and replicate by growth and fission.
• Peroxisomes lack their own DNA and ribosomes; all peroxisomal proteins are nuclear encoded, synthesised on free cytosolic ribosomes and imported post-translationally.
• At least 32 peroxisomal proteins (peroxins) are known that function in import and peroxisome biogenesis.
• Most peroxisomal matrix proteins carry a C-terminal PTS1 targeting signal; a minority use an N-terminal PTS2 signal; neither is typically cleaved after import.
• Cytosolic receptors recognise PTS1 or PTS2 and deliver cargo to a common import machinery on the peroxisomal membrane.
• Zellweger syndrome is an autosomal recessive disorder of peroxisome assembly in which import of many peroxisomal proteins is impaired, resulting in severe defects.

Key Points on Peroxisomes

• Peroxisomes are important for lipid metabolism and detoxification reactions.
• They catalyse oxidation reactions that break down fatty acids and some amino acids.
• Peroxisomes neutralise reactive oxygen species by converting H2O2 into water and oxygen.
• Liver cells, active in detoxification, contain abundant peroxisomes.

Nucleus

• The nucleus is the largest organelle in most eukaryotic cells and contains the cell's genetic material organised with histones into chromosomes.
• The nuclear envelope consists of inner and outer membranes separated by the perinuclear space and supported by the nuclear lamina.
• The outer nuclear membrane is continuous with the ER and is studded with ribosomes.
• Molecules such as proteins and RNAs cross the nuclear envelope via nuclear pore complexes rather than by membrane fusion.

Nuclear Targeting

• Nuclear pore complexes are large assemblies composed of about 100 different proteins that mediate selective transport between nucleus and cytoplasm.
• Typical NLS motifs are clusters of basic residues (for example PKKKRLV).
• Importins and exportins (karyopherins) mediate nuclear import and export respectively; the importin α/β heterodimer is a common nuclear import receptor.
• A gradient of Ran-GTP (high in the nucleus) versus Ran-GDP (high in the cytosol) provides directionality for nuclear transport.
• Ran-GEF in the nucleus generates Ran-GTP, and Ran-GAP in the cytosol stimulates GTP hydrolysis to Ran-GDP.
• NTF2 recycles Ran-GDP back into the nucleus to maintain the Ran gradient.

Nuclear Import

• Cargo proteins bearing an NLS bind importin in the cytoplasm to form a complex that interacts with the nuclear pore complex and translocates into the nucleus.
• Inside the nucleus Ran-GTP binds importin, causing a conformational change that releases the cargo.
• The importin-Ran-GTP complex returns to the cytoplasm, where Ran-GAP stimulates GTP hydrolysis and importin is released.
• Ran-GDP is returned to the nucleus by NTF2 and converted to Ran-GTP by Ran-GEF to restart the cycle.

Nuclear Export

• Exportins bind cargo in the nucleus together with Ran-GTP to form an export complex that translocates through the nuclear pore to the cytoplasm.
• In the cytoplasm Ran-GAP stimulates GTP hydrolysis on Ran, causing release of cargo from exportin.
• Ran-GDP is recycled back into the nucleus and converted to Ran-GTP by Ran-GEF to sustain export cycles.

Perspective for the Future

• Further genetic and biochemical studies in yeast and mammals will continue to clarify the mechanisms of protein translocation and organelle targeting.
• Improved understanding of translocation systems will deepen insight into cellular organisation and disease mechanisms.