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Protein targeting

Protein targeting, also known as protein sorting, is the essential cellular process by which newly synthesized proteins are directed from their site of synthesis on ribosomes to specific locations within the cell or for export outside it, ensuring proper function and compartmentalization within cells across all organisms.^[1] This process relies on inherent sorting signals embedded in the protein's amino acid sequence, which are recognized by dedicated receptor proteins and transport machinery to guide proteins to destinations such as the endoplasmic reticulum (ER), mitochondria, nucleus, peroxisomes, lysosomes, or the plasma membrane.^[1] Defects in protein targeting can lead to severe disorders, such as I-cell disease, a lysosomal storage disorder caused by mislocalization of enzymes due to mutations in targeting signals.^[2] In eukaryotic cells, protein targeting occurs primarily through two mechanisms: co-translational and post-translational translocation. Co-translational targeting involves proteins being translocated across or into membranes during their synthesis on ribosomes, most notably for secretory proteins and those destined for the ER, Golgi apparatus, lysosomes, or plasma membrane via the vesicular transport pathway.^[1] This process is mediated by the signal recognition particle (SRP), which binds to an N-terminal signal sequence—a short stretch of 15–30 hydrophobic amino acids—on the emerging polypeptide, pausing translation until the ribosome docks at the ER membrane's translocon.^[1] Post-translational targeting, in contrast, occurs after protein synthesis is complete in the cytosol and is used for import into organelles like mitochondria, chloroplasts (in plants), peroxisomes, and the nucleus, where proteins fold and are then actively transported through specific pores or channels.^[1] The specificity of targeting is determined by diverse signal sequences or signal patches, which vary by destination organelle and are often cleaved after import to allow proper folding. For instance, mitochondrial proteins feature an amphipathic N-terminal presequence with positively charged residues that directs them to translocases in the outer and inner membranes, while nuclear proteins contain nuclear localization signals (NLS)—short basic amino acid motifs like lysine or arginine clusters—that interact with importins for passage through nuclear pore complexes.^[1] Peroxisomal targeting signals (PTS) are typically C-terminal tripeptides, such as serine-lysine-leucine (SKL), recognized by cytosolic receptors like PEX5 for import.^[1] Vesicular transport in the endomembrane system further refines sorting, with proteins acquiring additional signals in the Golgi for delivery to lysosomes (e.g., mannose-6-phosphate tags) or secretion.^[1] Overall, these mechanisms maintain cellular homeostasis by preventing mislocalization, which could otherwise disrupt organelle function or trigger quality control degradation pathways.^[2]

Fundamentals

Definition and Cellular Importance

Protein targeting is the biological process by which newly synthesized proteins are directed from their sites of synthesis, typically ribosomes in the cytosol, to specific intracellular destinations such as organelles, membranes, or the extracellular space, utilizing dedicated signals and molecular machinery to ensure precise localization.^[3] This directed transport involves recognition of targeting sequences by chaperones and receptors, followed by translocation across or into membranes via specialized complexes.^[4] The cellular importance of protein targeting lies in its role in maintaining compartmentalization, which is essential for proper protein folding, functional assembly, and execution of specialized processes; for instance, it enables ATP production in mitochondria, lipid and protein synthesis in the endoplasmic reticulum (ER), and secretion of extracellular matrix components.^[5] Without accurate targeting, proteins may aggregate in the cytosol, undergo premature degradation, or engage in off-target interactions, leading to cellular dysfunction; studies indicate that mistargeting can affect 5–30% of translocated proteins in experimental models, underscoring the need for quality control mechanisms.^[6] In eukaryotes, roughly one-third of the proteome requires targeting to the ER for processing and export, while approximately 13% is directed to mitochondria, illustrating the process's broad impact on cellular proteome organization.^[5]^[7] These targeting mechanisms exhibit remarkable evolutionary conservation, originating in prokaryotes as basic translocation systems for membrane insertion and cargo transport, which were later adapted in eukaryotes to support endosymbiotic organelles like mitochondria and chloroplasts.^[3] This conservation reflects the fundamental role of spatial protein organization in life across domains, with eukaryotic innovations building upon prokaryotic precursors such as the Sec and Tat pathways.^[3]

Historical Milestones

In the 1950s and 1960s, George Palade's pioneering use of electron microscopy revealed the secretory pathway in eukaryotic cells, identifying the rough endoplasmic reticulum as the primary site for synthesizing secretory and membrane proteins, with their subsequent transport through the Golgi apparatus to secretory vesicles.^[8] This work established the foundational framework for understanding intracellular protein trafficking and earned Palade the Nobel Prize in Physiology or Medicine in 1974, shared with Albert Claude and Christian de Duve. A major breakthrough came in 1971 when Günter Blobel and David Sabatini proposed the signal hypothesis, suggesting that proteins destined for the secretory pathway or ER membrane contain an N-terminal signal peptide that directs their targeting during or after synthesis. Experimental validation followed, including the 1975 discovery by Blobel and Bernhard Dobberstein of the signal recognition particle (SRP), a ribonucleoprotein complex that binds the signal peptide on nascent polypeptides to facilitate co-translational targeting to the ER. Blobel's contributions culminated in his receipt of the Nobel Prize in Physiology or Medicine in 1999.^[9] Parallel developments in the 1970s and 1980s focused on organelle-specific targeting. Gottfried Schatz demonstrated that most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol, and imported post-translationally via N-terminal presequences, with his 1979 experiments confirming this for cytochrome c1. Schatz's group further identified components of the import machinery, including the TOM complex in the outer mitochondrial membrane through isolation of the 42 kDa ISP42 protein in 1989.^[10] For chloroplasts, Colin Robinson and colleagues in the 1980s characterized transit peptides as N-terminal targeting signals and purified processing proteases that cleave them upon import, as detailed in their 1984 study on pea chloroplast proteases.^[11] Post-2000 advances leveraged structural biology to visualize targeting mechanisms at atomic resolution. Cryo-electron microscopy (cryo-EM) structures of the Sec61 translocon, such as the 2014 mammalian ribosome-Sec61 complex at 3.4 Å resolution by Rebecca M. Voorhees et al., illuminated the channel's role in co- and post-translational translocation across the ER membrane.^[12] Concurrently, studies reinforced the essential function of Hsp70 chaperones in post-translational targeting, with 2006 experiments by M. Mokranjac et al. showing that individual Hsp70 molecules accelerate polypeptide unfolding and import into mitochondria without additional factors.^[13] More recent work includes the 2023 cryo-EM structure of the ribosome-Sec61 complex bound to the translocon-associated protein (TRAP) complex by Pavel Itskanov et al., providing further insights into accessory protein interactions during translocation.^[14]

Year	Scientist(s)	Breakthrough
1950s–1960s	George Palade	Elucidation of the secretory pathway via electron microscopy, identifying rough ER's role in protein synthesis and trafficking.^[8]
1971	Günter Blobel, David Sabatini	Proposal of the signal hypothesis for ER targeting via N-terminal signal peptides.
1975	Günter Blobel, Bernhard Dobberstein	Discovery of the signal recognition particle (SRP) for co-translational targeting.
1979	Gottfried Schatz	Demonstration of post-translational import of mitochondrial proteins like cytochrome c1.
1984	Colin Robinson et al.	Identification and purification of chloroplast transit peptide processing proteases.^[11]
1989	Gottfried Schatz et al.	Isolation of ISP42 as a key component of the mitochondrial outer membrane import complex (TOM).^[10]
2014	Rebecca M. Voorhees et al.	Cryo-EM structure of the mammalian ribosome-Sec61 translocon complex at 3.4 Å resolution.^[12]
2023	Pavel Itskanov et al.	Cryo-EM structure of the ribosome-Sec61 complex with the TRAP complex.^[14]

Targeting Signals

Signal Peptides and Sequences

Signal peptides, also known as leader peptides, are short N-terminal sequences typically comprising 15-30 amino acids that direct nascent proteins to specific cellular compartments, particularly the secretory pathway in eukaryotes and the export machinery in prokaryotes.^[15] These sequences exhibit a tripartite structure consisting of an N-region, H-region, and C-region. The N-region is a short, positively charged segment (1-5 residues) rich in basic amino acids such as lysine (K), arginine (R), and histidine (H), which initiates after the start methionine and imparts an overall positive charge to facilitate interaction with translocation components.^[15] The central H-region forms a hydrophobic core of 7-15 residues, predominantly composed of non-polar amino acids like leucine (L), isoleucine (I), and valine (V), enabling membrane insertion and targeting.^[15] The C-region, typically 3-7 residues long and polar or neutral, contains the cleavage site recognized by signal peptidases and is often shorter in eukaryotes compared to prokaryotes.^[15] Mitochondrial targeting presequences represent a specialized class of N-terminal signals, usually 20-80 amino acids in length, characterized by their ability to form amphipathic α-helices with one hydrophobic face and one positively charged face.80691-1.pdf) These presequences are enriched in positively charged residues (K/R) and hydroxylated amino acids (serine, threonine), while largely lacking acidic residues, contributing to their net positive charge and helical propensity. A common consensus motif within these presequences is φχχφφ, where φ denotes a hydrophobic residue and χ any amino acid, which supports their structural flexibility and targeting efficiency.^[16] Beyond N-terminal signals, protein targeting employs diverse internal or C-terminal sequences. Peroxisomal targeting signal 1 (PTS1) is an internal or C-terminal motif, most commonly the tripeptide serine-lysine-leucine (SKL) or close variants, embedded within a short sequence of about 12 amino acids that directs matrix proteins to peroxisomes.^[17] Nuclear localization signals (NLS) are typically internal basic clusters; the classic monopartite NLS from the SV40 large T antigen is the heptapeptide proline-lysine-lysine-lysine-arginine-lysine-valine (PKKKRKV), which mediates nuclear import through binding to importin α.^[18] For endoplasmic reticulum (ER) retention, soluble resident proteins bear a C-terminal tetrapeptide signal such as lysine-aspartate-glutamate-leucine (KDEL), which prevents escape to the Golgi by facilitating retrieval.^[19] The biophysical properties of these signals are critical for their function, with many exhibiting amphipathicity—combining hydrophobic and hydrophilic elements—to promote membrane association without disrupting bilayer integrity.^[15] The H-region of classical signal peptides and mitochondrial presequences often adopts an α-helical conformation in membrane-mimetic environments, enhancing translocation efficiency through stabilized interactions.^[15] Cleavage of these signals occurs post-targeting by specific peptidases; for ER-directed signal peptides, signal peptidase I (SPase I), a multi-subunit complex, performs endoproteolytic cleavage at the C-region site following the (−3, −1) rule (small residues like alanine at positions −3 and −1 relative to the cleavage point, e.g., Ala-X-Ala).^[20] Diversity in signal sequences exists across organisms, reflecting adaptations to distinct translocation systems. Bacterial signal peptides share the tripartite N-H-C structure with eukaryotic counterparts but are generally longer (especially in Gram-positive bacteria) and exhibit higher net positive charge in the N-region, with fewer modifications like glycosylation.^[15] In archaea, variants often feature twin-arginine (RR) motifs in the N-region for the twin-arginine translocation (TAT) pathway, enabling export of folded proteins across the plasma membrane while maintaining the hydrophobic H-core.^[21] Representative examples illustrate these signals' roles. The preproinsulin signal peptide, a 24-residue N-terminal sequence (MALWMRLLPL LALLALWGPD PAAA), directs the precursor to the ER for co-translational translocation and subsequent processing into mature insulin.^[22] For mitochondria, the presequence of cytochrome c oxidase subunit IV (COX4), a 25-residue N-terminal extension (MLSRLLRVSR LGSRRLLPVR ARLA), exemplifies the amphipathic helical motif that targets this nuclear-encoded subunit to the inner membrane after import.^[23]

Recognition by Chaperones and Receptors

Molecular chaperones such as Hsp70 and Hsp90 play crucial roles in protein targeting by preventing aggregation of nascent or unfolded polypeptides and maintaining them in competent states for recognition by downstream transport machinery.^[24] Hsp70 binds to exposed hydrophobic regions of client proteins in an ATP-dependent manner, stabilizing unfolded conformations that are essential for subsequent interactions with targeting receptors.^[25] Hsp90, often acting later in the folding pathway, collaborates with Hsp70 to remodel proteins and facilitate their delivery to specific organelles, such as mitochondria.^[26] Co-chaperones, including J-proteins (also known as DnaJ homologs), enhance the efficiency of these processes by stimulating the ATPase activity of Hsp70, which drives cycles of substrate binding and release.^[27] The signal recognition particle (SRP) serves as a key receptor complex for co-translational targeting to the endoplasmic reticulum (ER), recognizing signal peptides as they emerge from the ribosome. SRP's SRP54 subunit contains a methionine-rich M domain with a deep hydrophobic groove that accommodates the hydrophobic core of signal peptides through non-specific interactions, enabling broad specificity for diverse signal sequences.^[28] Upon binding, SRP pauses translation and docks to the SRP receptor on the ER membrane via reciprocal interactions between their GTPase domains, forming a stable SRP-receptor complex.^[29] This association is energy-dependent, with GTP hydrolysis by both GTPases triggering the release of the signal peptide to the translocon and dissociation of SRP for recycling.^[30] In mitochondrial protein import, specificity is achieved through distinct outer membrane receptors: TOM20 primarily recognizes N-terminal presequences via electrostatic interactions with their amphipathic helical structure, while TOM70 preferentially binds internal targeting signals of carrier proteins through hydrophobic contacts.^[31] Cytosolic chaperones deliver preproteins to these receptors; for instance, Hsp70 associates with presequence-containing proteins for handover to TOM20, whereas Hsp70 and Hsp90 cooperate to present carrier preproteins to TOM70.^[26] This dual-receptor system ensures efficient sorting, with TOM20 handling matrix-destined proteins and TOM70 managing metabolite transporters.^[32] Nuclear import relies on importins as receptors that bind nuclear localization signals (NLS) through electrostatic interactions between basic residues in the NLS and negatively charged grooves in importin-α's armadillo repeat domain.^[33] Classical monopartite or bipartite NLS motifs fit into this concave binding surface, promoting high-affinity recognition and formation of the importin-cargo complex for transport through nuclear pores.^[34] The ATPase cycles of Hsp70 and Hsp90 provide energetic support for maintaining NLS-bearing proteins in unfolded states prior to receptor engagement, ensuring timely delivery to the nucleus.^[25]

Translocation Processes

Co-translational Translocation

Co-translational translocation is a process in which protein synthesis on cytosolic ribosomes and the concurrent insertion or translocation of the nascent polypeptide across a biological membrane occur simultaneously, primarily targeting secretory and integral membrane proteins to the endoplasmic reticulum (ER) in eukaryotes. This mechanism ensures that hydrophobic regions of membrane proteins are shielded from the aqueous cytosol and that proteins destined for the secretory pathway are efficiently directed to the ER lumen or membrane. The process is initiated when a hydrophobic signal peptide emerges from the ribosomal exit tunnel, triggering recognition and targeting events that couple translation to membrane insertion.^[35] The key components include the signal recognition particle (SRP), which is a ribonucleoprotein complex that binds the emerging signal peptide, halting elongation to allow targeting; the SRP receptor (SR) on the ER membrane, which docks the ribosome-nascent chain (RNC) complex; and the Sec61 translocon, a heterotrimeric protein complex (Sec61α, β, and γ) that forms an aqueous pore in the ER membrane through which the polypeptide thread passes. During translocation, oligosaccharyltransferase (OST), associated with the Sec61 complex, catalyzes the en bloc addition of N-linked glycans to asparagine residues in the consensus sequence Asn-X-Ser/Thr on the translocating chain, marking it for further ER processing. Additionally, the ER luminal chaperone BiP, an Hsp70 homolog, binds to the incoming polypeptide via ATP-dependent cycles, acting as a molecular ratchet to prevent back-sliding and drive unidirectional translocation into the lumen.^[36] The process unfolds in distinct steps: upon emergence of the signal peptide (typically 15-30 residues long with a hydrophobic core), SRP binds it co-translationally, inducing a pause in translation by interacting with the elongation factor EF2 (or eEF2 in eukaryotes) to create a targeting window of about 30-60 seconds. The SRP-RNC complex then diffuses to the ER membrane, where it engages the SR via GTP hydrolysis, leading to handover of the signal peptide to the Sec61 channel; the ribosome directly associates with Sec61, resuming translation and threading the chain through the pore. As the chain elongates, the signal peptide is cleaved by the signal peptidase complex (SPC) within the translocon, allowing the mature protein to fold in the ER lumen with assistance from chaperones like BiP, while transmembrane domains partition laterally into the lipid bilayer if applicable.^[35] This pathway is highly conserved and dominant in eukaryotes for the majority of ER-targeted proteins, ensuring fidelity in the secretory pathway. In prokaryotes, an analogous system employs a simpler SRP (lacking some eukaryotic subunits) and the SecYEG translocon, facilitating co-translational insertion of inner membrane proteins into the plasma membrane, underscoring the evolutionary universality of SRP-dependent targeting across domains of life. BiP-mediated regulation in eukaryotes, through its ATP-driven binding and release, provides an additional layer of control to maintain translocation efficiency against potential retrotranslocation forces.^[37]^[36]

Post-translational Translocation

Post-translational translocation refers to the process by which fully synthesized proteins in the cytosol are imported into organelles such as mitochondria and peroxisomes, distinct from co-translational mechanisms that couple synthesis to membrane crossing. In this pathway, proteins are maintained in a translocation-competent state by cytosolic chaperones, such as Hsp70, which prevent aggregation and facilitate targeting to organelle receptors before threading through specialized translocons powered by ATP hydrolysis or membrane potential.^[38] This mechanism is essential for proteins destined for non-ribosomal compartments, allowing cytosolic maturation steps like folding intermediates or cofactor assembly prior to import. In mitochondria, post-translational import into the matrix primarily involves the TIM23 complex, a translocon in the inner membrane composed of core subunits Tim23 and Tim17, which forms a protein-conducting channel. Precursor proteins bearing N-terminal presequences are first recognized by outer membrane receptors of the TOM complex and transferred to TIM23 via accessory factors like Tim50. The process is driven by the proton motive force (Δψ) across the inner membrane, which electrophoretically pulls the positively charged presequence through the channel, while matrix-localized mtHsp70, in association with Tim44 and the PAM motor, uses ATP to unfold the protein and generate a pulling force through iterative binding and release cycles.^[38]^[39] Following translocation, the presequence is cleaved by the mitochondrial processing peptidase (MPP) in the matrix to yield the mature protein. A representative example is the import of matrix proteins such as the β-subunit of F1-ATPase, which follows this presequence pathway.^[38] For peroxisomes, post-translational import relies on peroxisomal targeting signals (PTS), particularly PTS1—a C-terminal tripeptide such as -SKL—that is recognized by the soluble receptor Pex5 in the cytosol. The Pex5-cargo complex docks at the peroxisomal membrane via the Pex13-Pex14 complex, forming a transient translocon pore approximately 9 nm in diameter that accommodates folded proteins or oligomers. Translocation occurs without unfolding, facilitated by a nuclear pore-like hydrogel meshwork in the membrane formed by Pex13's YG domain, which selectively permits diffusion of Pex5-bound cargo; post-import, Pex5 is monoubiquitinated and recycled by the AAA ATPases Pex1 and Pex6.^[40] This pathway's advantage lies in its ability to import pre-assembled protein complexes, preserving enzymatic activity and enabling rapid peroxisome function in oxidative metabolism. An example is PTS1-mediated import of enzymes like catalase, which folds in the cytosol before matrix entry.^[40] In prokaryotes and chloroplasts, the twin-arginine translocation (TAT) pathway exemplifies post-translational import of folded proteins across energy-transducing membranes, utilizing twin-arginine motifs in signal peptides recognized by Tat receptors. The TAT translocon, comprising TatA, TatB, and TatC, harnesses the proton motive force to drive translocation without ATP, allowing export of cofactored proteins like photosynthetic enzymes in chloroplasts.^[40] This mechanism underscores the versatility of post-translational translocation in maintaining folded states essential for function in diverse cellular contexts.

Eukaryotic Organelle Sorting

Mitochondrial Targeting Pathways

Mitochondrial targeting pathways in eukaryotes ensure the precise delivery of nuclear-encoded proteins to specific subcompartments of the organelle, including the outer membrane (OM), intermembrane space (IMS), inner membrane (IM), and matrix. These pathways primarily operate post-translationally, with most precursor proteins synthesized in the cytosol and recognized by receptors on the mitochondrial surface before translocation. The translocase of the outer membrane (TOM) complex serves as the universal entry gate for nearly all mitochondrial proteins, where Tom20 and Tom70 act as primary receptors for presequence-containing and carrier proteins, respectively, facilitating passage through the Tom40 channel.^[41] For beta-barrel proteins destined for the OM, such as porins, import via TOM is followed by handover to the sorting and assembly machinery (SAM) complex, which inserts these proteins into the lipid bilayer; Sam50 forms the core channel, with Mdm10 stabilizing the process.^[42] Sorting signals guide this compartmental specificity. Matrix-targeted proteins typically feature cleavable N-terminal presequences, which form amphipathic alpha-helices rich in positive charges, recognized by cytosolic chaperones like Hsp70 and mitochondrial receptors. In contrast, IM carrier proteins, such as adenine nucleotide translocators, possess internal targeting signals consisting of multiple transmembrane domains with moderate hydrophobicity, lacking cleavable presequences.^[41] Upon crossing the OM via TOM, IMS proteins can follow a stop-transfer mechanism, where hydrophobic segments halt translocation and promote lateral release into the IMS, or the oxidative folding pathway mediated by the MIA machinery. The latter involves Mia40, an oxidoreductase that captures incoming cysteine-rich precursors via mixed disulfide bonds, enabling their oxidative folding and trapping in the IMS; this is particularly crucial for twin CX3C or CX9C motif proteins like Cox17.^[43] For IM insertion, proteins diverge at the IM translocases. Presequence-containing proteins engage the TIM23 complex, where the presequence is driven across the IM by the electrochemical potential (Δψ) and pulled into the matrix by ATP-dependent action of mtHsp70 within the presequence translocase-associated motor (PAM) complex, often with Tim44 as a scaffold. Some TIM23 substrates with additional hydrophobic stop-transfer signals are sorted laterally into the IM via the TIM23-SORT subcomplex, involving Tim21 and Mgr2. Carrier proteins, meanwhile, are escorted by small TIM chaperones (Tim9-Tim10 or Tim8-Tim13) across the IMS to the TIM22 complex, where Δψ facilitates insertion of their multiple transmembrane helices into the IM.^[44] In the matrix, presequences are cleaved by mitochondrial processing peptidase (MPP), and nascent proteins achieve their native fold with assistance from chaperones like mtHsp70, Hsp60, and Hsp10, preventing aggregation.^[41] Quality control mechanisms monitor these pathways to handle mislocalized or unfolded proteins. The mitochondrial unfolded protein response (UPRmt) detects accumulation of mislocalized precursors in the cytosol or import defects, triggering transcriptional upregulation of chaperones and proteases via ATFS-1/ATF5 and the integrated stress response to restore proteostasis. Additionally, cytosolic surveillance by ubiquitin-proteasome systems degrades unimported precursors, while intramitochondrial proteases like LON and i-AAA degrade aberrant imports.^[45]

Chloroplast Targeting Pathways

In photosynthetic eukaryotes, the chloroplast proteome consists of approximately 3,000 proteins, with the vast majority—over 90%—encoded by the nuclear genome and synthesized in the cytosol as precursors bearing N-terminal transit peptides (TPs).^[46] These precursors are imported across the double-membrane envelope via coordinated action of the translocon at the outer envelope membrane of chloroplasts (TOC) and the translocon at the inner envelope membrane of chloroplasts (TIC).^[47] The TOC complex, comprising receptor components Toc159 and Toc34 for initial TP recognition and the β-barrel channel Toc75 as the protein-conducting pore, facilitates envelope crossing in an energy-dependent manner driven by GTP hydrolysis and ATP-powered chaperones. Upon translocation through the inner envelope via the TIC complex—primarily involving the channel-forming Tic110 and the scaffold protein Tic40—the precursors reach the stroma, where stromal Hsp70 chaperones prevent aggregation and drive unfolding for import.^[47] In the stroma, the transit peptides are cleaved by the stromal processing peptidase (SPP), releasing the mature protein for subsequent sorting or folding.^[47] This post-import processing ensures proper localization within the chloroplast, which is unique among eukaryotic organelles due to its additional internal thylakoid membrane system housing the photosynthetic apparatus. Intra-chloroplast sorting to the thylakoid lumen employs bipartite targeting signals, consisting of the cleaved TP followed by a thylakoid transfer domain resembling bacterial signal peptides.^[48] Lumenal proteins are routed via two distinct post-translational pathways: the Sec-dependent pathway (cpSec), which translocates unfolded precursors in an ATP-dependent manner, as seen with the oxygen-evolving complex protein OE33 (PsbO); and the twin-arginine translocation (TAT) pathway (cpTat), which imports fully folded proteins using the proton motive force across the thylakoid membrane, exemplified by OE17 (PsbQ) and OE23 (PsbP).^[48] These pathways reflect evolutionary conservation from bacterial ancestors, with cpTat uniquely suited for cofactor-bound proteins like plastocyanin.^[48] Proteins destined for the thylakoid membrane, such as light-harvesting chlorophyll a/b-binding proteins (LHCPs), utilize signal peptide-like sequences exposed after stromal cleavage, often in conjunction with the chloroplast signal recognition particle (cpSRP) pathway for co-translational integration.^[47] This SRP-dependent mechanism involves GTP hydrolysis to target hydrophobic precursors to the thylakoid, preventing aggregation in the aqueous stroma.^[47] A subset of nuclear-encoded proteins exhibits dual targeting to both chloroplasts and mitochondria, mediated by ambiguous N-terminal signals that share partial homology with mitochondrial targeting sequences and chloroplast TPs, allowing stochastic distribution based on chaperone interactions and import kinetics.^[49] Examples include the protease Lon1 and components of the glycine decarboxylase complex, which support metabolic coordination between these organelles without requiring alternative splicing or processing.^[50]^[51] This dual localization enhances efficiency in plant cells, where such proteins constitute a small but functionally significant fraction of the organellar proteomes.^[49]

Nuclear Import and Export

Nuclear import and export enable the selective trafficking of proteins and RNAs between the cytoplasm and nucleus in eukaryotic cells, mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope. These processes are essential for gene expression, cell signaling, and maintenance of nuclear integrity. Small molecules and proteins below approximately 40 kDa can passively diffuse through the NPC, while larger macromolecules require active, energy-dependent transport facilitated by soluble receptors known as karyopherins.^[52] The directionality of transport is driven by a Ran-GTP gradient, with high Ran-GTP concentrations in the nucleus maintained by the chromatin-bound guanine nucleotide exchange factor RCC1 and cytoplasmic GTPase-activating protein RanGAP1.^[53] The import mechanism begins with nuclear localization signals (NLSs) on cargo proteins being recognized by importin α, which binds to importin β to form a heterodimeric receptor complex that shields the cargo for translocation through the NPC.^[54] This complex docks to the NPC via interactions with nucleoporins and translocates bidirectionally across the pore, powered by Brownian motion, until nuclear Ran-GTP binds to importin β, dissociating the complex and releasing the cargo into the nucleoplasm.^[55] Importin β is then recycled to the cytoplasm bound to Ran-GTP, where GTP hydrolysis dissociates the pair, allowing reuse. This Ran-GTP gradient ensures unidirectional import by promoting cargo release only in the nucleus.^[56] In contrast, nuclear export relies on nuclear export signals (NESs), typically leucine-rich motifs, recognized by exportins such as CRM1 (also known as XPO1), which forms a ternary complex with the NES-cargo and Ran-GTP in the nucleus.^[57] This complex translocates through the NPC to the cytoplasm, where Ran-GTP hydrolysis, facilitated by RanGAP1 and RanBP1, releases the cargo and recycles the exportin. For RNA export, the NXF1 (TAP in humans) receptor, often with the NXT1 adaptor, mediates bulk mRNA export independently of Ran-GTP, binding mRNA via RNA-binding proteins and interacting with FG-nucleoporins. CRM1 handles export of proteins, tRNAs, and some mRNAs, ensuring precise spatiotemporal control.^[58] The NPC structure features a ~120 MDa scaffold with eightfold symmetry, comprising ~30 distinct nucleoporins (Nups), where FG-nucleoporins (FG-Nups) line the central channel to form a selective permeability barrier through hydrophobic interactions and phase separation into a hydrogel-like mesh.^[59] Karyopherin-cargo complexes transiently interact with FG repeats to partition through this barrier, enabling high throughput with each NPC supporting up to ~1,000 translocation events per second without compromising selectivity. Regulation of nuclear transport occurs via post-translational modifications, notably phosphorylation, which modulates NLS and NES accessibility or receptor affinity in a cell cycle-dependent manner. For instance, phosphorylation near the NLS of v-Jun inhibits importin binding during interphase, restricting nuclear entry until mitosis, while dephosphorylation activates import to coordinate oncogenic signaling.^[60] Such controls link transport to cell cycle progression, ensuring proteins like cyclins enter the nucleus at appropriate phases.^[61]

Endoplasmic Reticulum Targeting

Proteins destined for the secretory pathway or for insertion into the endoplasmic reticulum (ER) membrane are primarily targeted co-translationally. Nascent polypeptides bearing an N-terminal signal peptide are recognized by the signal recognition particle (SRP), a ribonucleoprotein complex that binds to the signal sequence as it emerges from the ribosome. This interaction pauses translation and directs the ribosome-nascent chain complex to the ER membrane via docking to the SRP receptor (SR). The SRP then facilitates transfer of the signal peptide to the Sec61 translocon, a heterotrimeric protein channel composed of Sec61α, Sec61β, and Sec61γ subunits, which serves as the primary conduit for protein translocation across or into the ER membrane.^[62]^[63] Co-translational insertion ensures that hydrophobic transmembrane domains are shielded from the cytosol during membrane integration, minimizing aggregation risks.^[62] Once in the ER lumen or membrane, proteins are subject to retention mechanisms to prevent unintended export. Soluble luminal proteins, such as chaperones, contain a C-terminal KDEL sequence (Lys-Asp-Glu-Leu) that binds to KDEL receptors in the cis-Golgi, triggering retrograde transport back to the ER via COPI vesicles.^[64] For type I membrane proteins, a dilysine motif (KKXX, where X is any amino acid) in the C-terminal cytoplasmic tail interacts with coat protein I (COPI) components, similarly mediating retrieval from the Golgi.^[64] These signals ensure that ER-resident proteins maintain their localization, with the KDEL receptor's affinity modulated by pH and calcium gradients between compartments.^[65] Quality control in the ER involves chaperone-assisted folding and degradation pathways. The calnexin/calreticulin cycle regulates glycoprotein folding: upon translocation, N-linked glycans are added to asparagine residues in the consensus sequence Asn-X-Ser/Thr by oligosaccharyltransferase (OST), associated with the Sec61 translocon.^[66] Glucosidase I and II trim the outermost glucose residues, allowing binding to the lectin chaperones calnexin (membrane-bound) or calreticulin (luminal), which retain the glycoprotein and recruit folding enzymes like UDP-glucose:glycoprotein glucosyltransferase (UGGT) to reglucosylate misfolded proteins for repeated cycles.^[67] Misfolded or unassembled proteins are directed to ER-associated degradation (ERAD), where they are retrotranslocated through Sec61 or other channels into the cytosol, polyubiquitinated by E3 ligases such as Hrd1 or Doa10, and degraded by the 26S proteasome.^[68] This ubiquitin-proteasome pathway eliminates terminally misfolded proteins, maintaining ER proteostasis.^[69] Properly folded proteins exit the ER via COPII-coated vesicles budding from ER exit sites (ERES). The GTPase Sar1 recruits the Sec23/24-Sec13/31 coat complex to the membrane, where Sec24 acts as a cargo adaptor selecting soluble and membrane proteins with specific export motifs, such as dileucine or diacidic sequences.^[70] Cargo receptors like Erv29 further enhance packaging of secretory proteins into these 60-80 nm vesicles, which fuse with the cis-Golgi or intermediate compartments to initiate anterograde transport.^[71] This selective packaging ensures efficient sorting while excluding ER residents.^[70]

Peroxisomal Targeting

Peroxisomal targeting enables the selective import of proteins into peroxisomes, single-membrane-bound organelles specialized for oxidative reactions such as fatty acid beta-oxidation and reactive oxygen species detoxification. Unlike many other organelles, peroxisomes import fully folded proteins, oligomeric complexes, and even those bound to cofactors, without requiring protein unfolding prior to translocation across the membrane. This post-translational process relies on specific targeting signals and receptor-mediated shuttling, allowing peroxisomes to assemble functional enzyme complexes rapidly in response to cellular needs.^[72] The majority of peroxisomal matrix proteins contain a peroxisomal targeting signal type 1 (PTS1), a short C-terminal tripeptide sequence with the consensus motif serine-lysine-leucine (SKL) or close variants such as serine-lysine-methionine (SKM). This signal is recognized in the cytosol by the soluble receptor protein PEX5, which binds the PTS1 motif via tetratricopeptide repeat domains in its C-terminal region. A smaller group of proteins, primarily involved in early biosynthetic pathways, utilize the peroxisomal targeting signal type 2 (PTS2), an N-terminal nonapeptide with the consensus sequence arginine/leucine-x5-histidine/leucine (RLx5HL), where x represents any amino acid. The PTS2 is specifically bound by the receptor PEX7, which forms a complex with PEX5 to facilitate import. These signals ensure precise sorting, with PTS1 directing over 90% of matrix proteins and PTS2 handling the rest.^[73] Once bound to their receptors, cargo proteins are shuttled to the peroxisomal membrane, where PEX5 (for PTS1) or the PEX5-PEX7 complex (for PTS2) docks via interactions with the membrane-anchored peroxins PEX13 and PEX14, forming a transient import pore. Cargo release occurs inside the peroxisome, after which the receptors are monoubiquitinated at a conserved cysteine residue by the RING finger E3 ubiquitin ligase complex composed of PEX2, PEX10, and PEX12. This ubiquitination marks the receptors for extraction back to the cytosol by the AAA ATPase peroxins PEX1 and PEX6, enabling receptor recycling and preventing their accumulation on the membrane. The tolerance for folded structures is exemplified by the import of catalase, a tetrameric enzyme with bound heme cofactors, which assembles in the cytosol before translocation.^[74]^[75] Peroxisomes arise de novo from pre-peroxisomal vesicles derived from the endoplasmic reticulum membrane, which bud off and fuse with additional precursors to form mature organelles. Each peroxisome typically incorporates around 50 distinct matrix proteins, reflecting their compact proteome tailored to metabolic functions.^[76] This biogenesis pathway supports dynamic peroxisome proliferation in response to lipid metabolism demands.^[76]

Prokaryotic and Archaeal Targeting

Gram-negative Bacterial Systems

Gram-negative bacteria possess a complex cell envelope consisting of an inner cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane, necessitating specialized mechanisms for protein targeting to the periplasm and outer membrane. Protein export primarily occurs via the Sec and TAT pathways across the inner membrane, followed by further sorting to the outer membrane or retention in the periplasm. These systems ensure the proper localization of enzymes, structural components, and virulence factors essential for bacterial survival and pathogenesis.^[77] The Sec pathway is the predominant route for exporting unfolded or partially folded proteins across the inner membrane to the periplasm in Gram-negative bacteria such as Escherichia coli. It utilizes the heterotrimeric SecYEG translocon embedded in the inner membrane, where SecY forms the protein-conducting channel, SecE stabilizes the complex, and SecG facilitates translocation cycles. This pathway supports both co-translational translocation, guided by the signal recognition particle (SRP) and FtsY receptor, and post-translational translocation, involving the chaperone SecB to prevent premature folding. Sec signal peptides, typically 18–30 amino acids long, feature a positively charged N-region, a hydrophobic core H-region, and a polar C-region with an AXA cleavage motif recognized by signal peptidase I for removal upon export. The energy for Sec-mediated translocation is provided by the ATPase activity of SecA, a peripheral membrane motor protein that undergoes cyclic ATP hydrolysis to drive stepwise substrate threading through the SecYEG channel, with each cycle advancing approximately 4–7 residues.^[78]^[79]^[80] In contrast, the twin-arginine translocation (TAT) pathway exports fully folded proteins across the inner membrane, particularly those requiring periplasmic cofactors like redox enzymes (e.g., copper amine oxidase or hydrogenase subunits). The TAT system in Gram-negative bacteria comprises TatA, TatB, and TatC, forming a receptor complex where TatB and TatC recognize the substrate, and multiple TatA protomers assemble into a dynamic pore for translocation. TAT signal peptides resemble Sec signals but include a conserved twin-arginine motif (S/T-R-R-x-F-L-K) in the H-region, which confers specificity and ensures folded protein compatibility. Unlike the Sec pathway, TAT translocation is powered exclusively by the proton motive force (PMF) across the inner membrane, with the transmembrane electrochemical gradient (Δψ component) driving the process without ATP hydrolysis. This PMF-dependent mechanism allows TAT to function under varying metabolic conditions, though it is sensitive to dissipation of the gradient.^[81]^[82]^[83] Once in the periplasm, proteins destined for the outer membrane are targeted via chaperone-mediated pathways. Beta-barrel outer membrane proteins (OMPs), such as porins, are escorted by periplasmic chaperones like SurA and Skp to the beta-barrel assembly machinery (BAM) complex, a five-subunit system (BamA–E) anchored in the outer membrane. BamA, an OMP with a β-barrel domain and periplasmic POTRA repeats, catalyzes the insertion and folding of substrate barrels in an ATP-independent manner, leveraging lateral opening of its barrel for substrate handover. The Lpt (lipopolysaccharide transport) system, involving seven proteins (LptA–G), bridges the periplasm to transport lipopolysaccharide (LPS) from the inner membrane to the outer leaflet of the outer membrane, with LptB2FGC forming an ABC transporter at the inner membrane and LptCDE inserting LPS via a periplasmic LptA polymer. While primarily for LPS, the Lpt machinery integrates with OMP biogenesis to maintain outer membrane asymmetry and integrity. These outer membrane targeting steps highlight the coordinated protein-protein interactions essential for Gram-negative envelope assembly.00375-2)^[84]^[85]

Gram-positive Bacterial Systems

In Gram-positive bacteria, protein targeting primarily involves translocation across a single cytoplasmic membrane, lacking the outer membrane characteristic of Gram-negatives, which allows direct release of secreted proteins into the extracellular environment. The general secretion (Sec) pathway dominates this process, facilitating the export of unfolded proteins via the SecYEG translocon in a manner analogous to other bacteria, driven by the ATPase SecA and guided by N-terminal signal peptides. This pathway handles the majority of secretory proteins, including those destined for the cell wall or extracellular space, and is essential for processes such as nutrient acquisition, cell wall maintenance, and virulence factor deployment.^[86]^[87]^[88] For membrane protein insertion, Gram-positive bacteria rely on the YidC insertase, a conserved chaperone that operates independently or in cooperation with the Sec machinery to integrate transmembrane helices into the lipid bilayer. In model organisms like Bacillus subtilis, YidC homologs such as SpoIIIJ (YidC1) and YqjG (YidC2) support the biogenesis of respiratory chain complexes and other essential membrane proteins, with either paralog sufficient for viability, though double mutants are lethal. Alternative pathways expand targeting capabilities; for instance, the ESX (Type VII) secretion system in actinobacteria like Mycobacterium tuberculosis exports folded proteins, including virulence factors such as ESAT-6 and CFP-10, across the membrane using a specialized ATPase-driven apparatus without classical signal peptides. Additionally, the phage shock protein (Psp) response, present in some Gram-positives like Streptococcus pneumoniae, mitigates envelope stress that could impair targeting by stabilizing the membrane during protein translocation overload.^[89]^[90]^[91]^[92] Specific targeting signals direct proteins to distinct fates. Lipoproteins, which anchor in the outer leaflet of the cytoplasmic membrane, are recognized by a conserved lipobox motif (L-[S/T/A/V]-[A/G]-C) in their signal peptide, enabling acylation at the cysteine residue by prolipoprotein diacylglyceryl transferase (Lgt) followed by cleavage and sorting. This pathway is crucial for envelope integrity and immune evasion in pathogens. For extracellular localization, sortase enzymes covalently anchor surface proteins to peptidoglycan via an LPXTG sorting motif, with sortase A in Staphylococcus aureus and B. subtilis linking pilins, adhesins, and enzymes to the cell wall, enhancing host colonization. In B. subtilis, the Sec secretome comprises around 200-300 proteins, including hydrolases like subtilisin and proteases, which constitute up to 25% of total cellular protein under optimal growth conditions, underscoring the pathway's role in industrial enzyme production and environmental adaptation.^[93]^[94]^[95]^[96]^[97]^[98]

Archaeal Mechanisms

Archaea employ a combination of protein targeting mechanisms that exhibit both prokaryotic and eukaryotic characteristics, facilitating the export of proteins across the cytoplasmic membrane and their integration into the membrane. These systems are adapted to diverse environments, including extreme conditions, and primarily involve the Sec pathway for unfolded proteins and a signal recognition particle (SRP) system reminiscent of eukaryotes for co-translational targeting. Additional pathways, such as twin-arginine translocation (TAT)-like systems, enable the export of folded proteins in certain lineages.^[99] The Sec pathway in archaea utilizes homologs of the bacterial SecYE translocon, consisting of SecY and SecE, to mediate the translocation of secretory and membrane proteins across or into the cytoplasmic membrane. Unlike bacteria, archaea lack a SecA ATPase homolog, relying instead on ribosomal stalling during co-translational translocation or possibly ion gradients for post-translational export, with SecDF aiding in later stages. This pathway processes proteins bearing N-terminal signal peptides, which are cleaved upon translocation, and is essential for viability, as demonstrated by the ability of archaeal SecY to complement bacterial mutants.^[99]^[100]^[101] The SRP system in archaea closely mirrors the eukaryotic version, featuring a heterodimeric SRP composed of SRP54 (also called Ffh) and SRP19 bound to a 7S RNA, which recognizes hydrophobic signal sequences or transmembrane domains on nascent polypeptides emerging from the ribosome. This leads to translational pausing and targeting of the ribosome-nascent chain complex to the Sec translocon via interaction with the SRP receptor FtsY, a GTPase homologous to eukaryotic SRα. SRP54 is essential for cell viability and membrane protein biogenesis, with reconstitution studies in Haloferax volcanii confirming its co-translational role.^[99]^[101]^[100] Some archaeal species possess a TAT-like system for exporting fully folded proteins, utilizing twin-arginine motifs in N-terminal signal peptides and core components TatA and TatC, but lacking TatB found in bacteria. This pathway predominates in haloarchaea, where over 90% of secreted proteins in Halobacterium sp. NRC-1 are TAT-dependent, powered by the sodium motive force rather than proton motive force, facilitating rapid folding in high-salt environments. Examples include the halocin HalH4 in Haloferax mediterranei.^[99]^[101] Archaeal membrane proteins are predominantly α-helical and follow the "positive inside rule" for topology, with insertion often coordinated by the SRP-Sec system. Signal peptidases in archaea, such as type I homologs (e.g., Sec11), exhibit catalytic mechanisms more akin to eukaryotic signal peptidase complexes than bacterial ones, lacking conserved bacterial residues but sharing Ser-Lys dyads for cleavage of signal peptides post-translocation. These enzymes, present in duplicates like Sec11a and Sec11b in Haloferax volcanii, play distinct roles in processing secretory and membrane proteins.^[99]^[102]^[103] In thermophilic archaea, such as Pyrococcus furiosus, protein targeting is supported by heat-stable chaperones that prevent aggregation under high temperatures, including group II chaperonins and small heat shock proteins (sHSPs). These chaperones, like the exceptionally stable Cpn from P. furiosus, assist in folding and maintaining translocation-competent states for hyperthermophilic enzymes, such as α-amylase, without ATP-dependent motors like SecA. The heat shock response in P. furiosus upregulates these chaperones, enhancing secretion of surface proteins like flagellar components for motility in extreme heat.^[104]^[105]

Pathological Implications

Diseases Linked to Targeting Defects

Defects in mitochondrial protein targeting contribute to several mitochondrial disorders, primarily through disruptions in the import and assembly of nuclear-encoded proteins into the respiratory chain complexes. For instance, recessive forms of Leber's hereditary optic neuropathy (LHON) have been linked to biallelic mutations in DNAJC30, a cytosolic chaperone involved in complex I biogenesis, which impairs the import and assembly of nuclear-encoded subunits, leading to optic nerve degeneration and vision loss.^[106] Mutations in translocase components, such as TIMM50 in the TIM23 complex, disrupt preprotein import across the inner mitochondrial membrane, causing severe mitochondrial disorders with encephalopathy, lactic acidosis, and muscle weakness.^[107] Endoplasmic reticulum (ER) targeting failures often trigger ER stress and the unfolded protein response (UPR), contributing to diseases like cystic fibrosis and lysosomal storage disorders. In cystic fibrosis, the most common mutation, ΔF508 in the CFTR gene, causes the CFTR protein to misfold during ER translocation, leading to its recognition by ER-associated degradation (ERAD) machinery and subsequent ubiquitination and proteasomal destruction, preventing proper trafficking to the plasma membrane and resulting in impaired chloride transport.^[108] This ER retention and degradation pathway is a central mechanism in the disease's pathogenesis. A classic example of lysosomal targeting defect is I-cell disease (mucolipidosis II), caused by mutations in the GNPTAB gene encoding GlcNAc-1-phosphotransferase, which fails to add mannose-6-phosphate tags to lysosomal enzymes in the Golgi, resulting in their secretion instead of lysosomal delivery, accumulation of undegraded substrates, and severe multisystem involvement including skeletal dysplasia and developmental delay.^[2] Peroxisomal targeting defects underlie severe disorders such as Zellweger spectrum disorder (ZSD), characterized by mutations in PEX genes that encode peroxins essential for peroxisome biogenesis and protein import. Mutations in PEX1, the most common cause affecting nearly two-thirds of cases, disrupt the receptor recycling and translocation of peroxisomal targeting signal (PTS1 and PTS2) proteins across the peroxisomal membrane, leading to absent or dysfunctional peroxisomes, accumulation of very long-chain fatty acids, and multisystem failure including hypotonia, seizures, and liver dysfunction.^[109] These import blocks prevent the assembly of peroxisomal enzymes critical for lipid metabolism. Nuclear protein targeting impairments are implicated in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, TDP-43 mislocalization from the nucleus to the cytoplasm is a hallmark pathology, often driven by defects in nuclear import receptors such as importin β, which fail to efficiently transport TDP-43 via its nuclear localization signal, resulting in cytoplasmic aggregation, RNA processing disruptions, and motor neuron death.^[110] This mislocalization exacerbates neuronal toxicity and is observed in over 95% of sporadic ALS cases. In prokaryotes, disruptions in bacterial protein targeting pathways can be exploited for therapeutic purposes in infections. Inhibitors targeting SecA, the ATPase motor of the Sec translocase system in Gram-negative bacteria like Escherichia coli, block the post-translational translocation of secretory proteins across the cytoplasmic membrane, leading to protein export failure and bacterial lethality. Compounds such as sodium azide and pyrazolopyrimidinone derivatives have been identified as SecA inhibitors with antibacterial potential, highlighting the Sec pathway as a target for novel antibiotics against multidrug-resistant pathogens.

Therapeutic Targeting Strategies

Therapeutic targeting of protein targeting pathways has emerged as a promising strategy for treating disorders arising from defective protein localization, with modulators designed to enhance folding, import, or translocation efficiency. In the endoplasmic reticulum (ER), chemical chaperones such as 4-phenylbutyrate (4-PBA) promote proper folding of mislocalized proteins by reducing ER stress and facilitating secretion in conditions involving protein misfolding.^[111] For instance, 4-PBA has been shown to restore trafficking and increase secretion of mutant proteins in cellular models of folding diseases.^[112] Additionally, small-molecule proteostasis regulators reprogram ER stress responses to improve the folding and trafficking of secretory proteins, targeting nearly one-third of the proteome that enters the ER.^[113] These regulators act by modulating the unfolded protein response, thereby alleviating proteotoxic stress and enhancing overall ER homeostasis.^[114] For mitochondrial targeting defects, antioxidants mitigate oxidative damage that impairs mitochondrial function. Mitochondria-targeted antioxidants, such as ubiquinol derivatives like MitoQ, inhibit lipid peroxidation and offer therapeutic potential in degenerative diseases linked to mitochondrial dysfunction.^[115] Gene therapy approaches address mutations in nuclear-encoded mitochondrial proteins by delivering corrected genes via adeno-associated viral vectors, restoring function in preclinical models of mitochondrial disorders.^[116] Pathogenic variants in TIM23 genes, such as those affecting Tim50, disrupt preprotein translocation, and targeted gene replacement has shown promise in compensating for these defects without off-target effects on heteroplasmy.^[107] In peroxisomal disorders like Zellweger syndrome, therapies focus on stabilizing import receptors to rescue biogenesis. Long-term cholic acid administration sustains peroxisomal function by enhancing bile acid metabolism and preventing liver progression in patients with peroxisome assembly defects.^[117] Nitric oxide donors, such as S-nitrosoglutathione, promote peroxisome number and activity in PEX1 mutant fibroblasts, extending lifespan in model organisms by stabilizing import pathways.^[118] Antimicrobial strategies exploit bacterial protein targeting for pathogen control. Inhibitors of the Sec translocon, including small molecules targeting SecA ATPase activity, block post-translational export in Gram-negative bacteria, abrogating efflux pump function and enhancing antibiotic efficacy.^[119] For the twin-arginine translocation (TAT) pathway, novel small-molecule inhibitors disrupt folded protein export in pathogens like Campylobacter jejuni, reducing virulence and biofilm formation without mammalian toxicity.^[120] These TAT blockers, identified through high-throughput screens, synergize with existing antimicrobials to combat multidrug-resistant strains.^[121] Emerging therapies leverage gene editing and biomimetic delivery to modulate targeting signals. CRISPR-Cas9 systems enable precise correction of mutations in mitochondrial targeting sequences, shifting heteroplasmy and restoring import in cellular models of mitochondrial disease.^[122] Base editors adapted for mitochondrial DNA achieve efficient editing of import-related genes, modeling and alleviating pathogenic variants in rodents.^[123] Nanoparticle systems mimicking signal recognition particle (SRP)-mediated cotranslational targeting facilitate site-specific delivery of therapeutic proteins, enhancing cellular uptake and secretion in vivo.^[124] These engineered nanoparticles promote chain elongation arrest and microsomal docking, akin to SRP function, for precise protein production at target sites.^[125]

Computational Approaches

Prediction Algorithms

Prediction algorithms for protein targeting employ bioinformatics approaches to computationally identify targeting signals and predict subcellular destinations based on amino acid sequences. These tools analyze features such as N-terminal signal peptides, transit peptides, and other motifs to classify proteins into categories like secretory, mitochondrial, or chloroplastic pathways. Seminal methods rely on machine learning techniques, including neural networks and hidden Markov models, to achieve high predictive accuracy, often exceeding 90% for well-defined signals.^[126] One of the foundational tools is SignalP, which uses neural networks to predict the presence and cleavage sites of signal peptides in eukaryotic and prokaryotic proteins. Developed initially in the 1990s and iteratively improved, the latest version, SignalP 6.0, incorporates deep learning to distinguish all five types of signal peptides, including those for the twin-arginine translocation pathway, with accuracies surpassing 95% on benchmark datasets for cleavage site prediction.^[126] SignalP excels in identifying classical secretory signals but focuses primarily on N-terminal regions, making it essential for distinguishing exported proteins from cytosolic ones.^[127] TargetP complements SignalP by discriminating between mitochondrial, chloroplastic, secretory, and other targeting signals using deep neural networks trained on sequence motifs of variable lengths. The tool, in its second iteration (TargetP 2.0), predicts N-terminal presequences and their cleavage sites, achieving balanced accuracies around 85-90% across eukaryotic localizations by leveraging neural architectures suited for motif recognition. It is particularly useful for plant and animal proteins, integrating predictions to resolve overlaps between pathways like mitochondrial and secretory targeting. PSORT, including variants like PSORTb for bacteria and WoLF PSORT for eukaryotes, adopts a rule-based approach combined with machine learning to predict subcellular localization by integrating multiple sequence features such as amino acid composition, functional motifs, and known sorting signals. PSORTb 3.0, for instance, employs support vector machines on bacterial datasets to achieve precision rates of about 90% for outer membrane and periplasmic predictions in Gram-negative bacteria.^[128] This method's strength lies in its interpretability, allowing users to trace decisions back to specific rules, though it may underperform on novel or atypical sequences compared to purely data-driven models.^[129] Advancements in the 2020s have introduced deep learning models like DeepLoc, which utilize transformer-based protein language models and attention mechanisms to predict multi-label subcellular localizations directly from full protein sequences, bypassing explicit signal extraction. DeepLoc 2.0, for example, employs protein language models for eukaryotic predictions, attaining top-1 accuracies around 74% and F1 scores over 0.6 for 10 compartments while providing attention-based interpretability for key sequence regions.^[130] These models represent a shift toward end-to-end learning, improving handling of non-canonical signals and integrating evolutionary information for broader applicability across organisms.^[131] Recent developments have integrated structural predictions from tools like AlphaFold to refine targeting signal identification. For instance, analyses of AlphaFold2-predicted structures have improved the precision of TargetP 2.0 and SignalP 6.0 by confirming signal peptide conformations, reducing false positives in ambiguous cases by up to 20% on benchmark datasets as of 2023.^[132] Despite their efficacy, prediction algorithms face limitations in resolving ambiguous or weak targeting signals, such as those in dual-localized proteins or non-classical pathways, where false positives can exceed 10-20% in challenging cases. Additionally, reliance on training data biases toward well-studied organisms can reduce performance on under-represented species, underscoring the need for experimental validation to confirm in silico predictions.

Experimental Validation Tools

Fluorescence microscopy serves as a primary tool for visualizing protein localization in living cells, often employing fusions with green fluorescent protein (GFP) or its variants to track targeting signals. For instance, mito-GFP fusions have been widely used to confirm mitochondrial import by observing punctate fluorescence patterns colocalizing with mitochondrial markers like MitoTracker. This approach allows real-time monitoring of dynamic targeting processes, such as translocation to the endoplasmic reticulum or nucleus, with validation through colocalization coefficients exceeding 0.8 in fixed and live-cell imaging. However, potential artifacts from fusion-induced mislocalization necessitate controls like antibody staining for endogenous proteins.^[133]^[134] Subcellular fractionation, combined with differential centrifugation, enables the isolation of organelles to biochemically assess protein distribution. Cells are lysed and subjected to sequential centrifugation steps—typically low-speed (e.g., 1,000 × g) for nuclei, medium-speed (10,000 × g) for mitochondria, and high-speed (100,000 × g) for microsomes—followed by Western blotting with compartment-specific markers like COX IV for mitochondria or calnexin for ER. This method has quantified targeting efficiency, revealing, for example, over 70% enrichment of matrix proteins in mitochondrial fractions from yeast extracts. Protease protection assays on fractions further confirm import by assessing resistance to added proteases, distinguishing surface-bound from translocated proteins.^[135]^[136] Chemical cross-linking techniques identify transient interactions between targeting factors and cargo proteins, such as the signal recognition particle (SRP) with its receptor. UV- or chemical-induced cross-linking (e.g., using DSS or BS3) captures SRP54-signal sequence complexes in translationally active lysates, followed by immunoprecipitation and mass spectrometry or SDS-PAGE analysis. Seminal studies have mapped SRP-receptor interfaces, showing cross-linked adducts at residues critical for GTPase activation, with efficiencies up to 50% in reconstituted systems. This approach is particularly useful for prokaryotic and eukaryotic co-translational targeting pathways.^[137]^[138] In vitro import assays reconstitute targeting using radiolabeled precursor proteins synthesized via in vitro transcription-translation and isolated organelles. For mitochondrial import, [³⁵S]-methionine-labeled preproteins are incubated with energized mitochondria under ATP-dependent conditions, with import scored by protease-protected, alkali-resistant integration (typically 20-60% efficiency for matrix proteins). This system has elucidated receptor dependencies, such as TOM20/TOM70 roles, by blocking with antibodies and quantifying via SDS-PAGE autoradiography. Adaptations to human cell mitochondria maintain physiological relevance for studying import defects.^[139]^[140] Proteomics approaches, leveraging mass spectrometry, detect targeting defects in mutants by profiling proteome-wide changes in localization. Label-free or TMT-based quantitative MS on fractionated samples identifies mislocalized proteins, such as accumulation of precursors in cytosolic fractions of import mutants (e.g., tom40Δ yeast strains showing 2-5-fold upregulation of non-imported preproteins). Interactome analysis via cross-linking MS maps translocation complexes, revealing novel substrates for pathways like MIA40-dependent import. These methods have scaled to genome-wide screens, prioritizing high-confidence hits with spectral counts >10.^[141]^[142]

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Identification and evaluation of twin-arginine translocase inhibitors
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