@article{Jeong2025,

title = {Intracellular delivery of proteins for live cell imaging},

author = {Ban-Seok Jeong and Hwanhee C. Kim and Catherine M. Sniezek and Stephanie Berger and Justin M. Kollman and David Baker and Joshua C. Vaughan and Xiaohu Gao},

url = {https://www.sciencedirect.com/science/article/pii/S0168365925002718, Journal of Controlled Release

https://www.bakerlab.org/wp-content/uploads/2025/03/1-s2.0-S0168365925002718-main.pdf, PDF},

doi = {10.1016/j.jconrel.2025.113651},

year  = {2025},

date = {2025-03-28},

urldate = {2025-05-00},

journal = {Journal of Controlled Release},

publisher = {Elsevier BV},

abstract = {The majority of cellular functions are regulated by intracellular proteins, and regulating their interactions can unlock fundamental insights in biology and open new avenues for drug discovery. Because the vast majority of intracellular targets remain undruggable, there is significant current interest in developing protein-based agents especially monoclonal antibodies due to their specificity, availability, and established screening/engineering methods. However, efficient delivery of proteins into the cytoplasm has been a major challenge in biological engineering and drug discovery. We previously reported a platform technology based on a Coomassie blue-cholesterol conjugate (CB-tag) capable of delivering small proteins directly into the cytoplasm. Here, we report a new generation of CB-tag that can bring proteins with a wide size range into the cytoplasm, bypassing endosomal sequestration. Remarkably, intracellular targets with distinct structures were visualized. Overall, the new CB-tag demonstrated a robust ability in protein delivery with broad applications ranging from live-cell immunofluorescence to protein-based therapeutic development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lee2024b,

title = {Four-component protein nanocages designed by programmed symmetry breaking},

author = {Sangmin Lee and Ryan D. Kibler and Green Ahn and Yang Hsia and Andrew J. Borst and Annika Philomin and Madison A. Kennedy and Buwei Huang and Barry Stoddard and David Baker},

url = {https://www.nature.com/articles/s41586-024-07814-1, Nature [Open Access]},

doi = {10.1038/s41586-024-07814-1},

year  = {2024},

date = {2024-12-18},

urldate = {2024-12-18},

journal = {Nature},

publisher = {Springer Science and Business Media LLC},

abstract = {Four, eight or twenty C3 symmetric protein trimers can be arranged with tetrahedral, octahedral or icosahedral point group symmetry to generate closed cage-like structures1,2. Viruses access more complex higher triangulation number icosahedral architectures by breaking perfect point group symmetry3,4,5,6,7,8,9, but nature appears not to have explored similar symmetry breaking for tetrahedral or octahedral symmetries. Here we describe a general design strategy for building higher triangulation number architectures starting from regular polyhedra through pseudosymmetrization of trimeric building blocks. Electron microscopy confirms the structures of T = 4 cages with 48 (tetrahedral), 96 (octahedral) and 240 (icosahedral) subunits, each with 4 distinct chains and 6 different protein–protein interfaces, and diameters of 33 nm, 43 nm and 75 nm, respectively. Higher triangulation number viruses possess very sophisticated functionalities; our general route to higher triangulation number nanocages should similarly enable a next generation of multiple antigen-displaying vaccine candidates10,11 and targeted delivery vehicles12,13.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Pillai2024,

title = {De novo design of allosterically switchable protein assemblies},

author = {Arvind Pillai and Abbas Idris and Annika Philomin and Connor Weidle and Rebecca Skotheim and Philip J. Y. Leung and Adam Broerman and Cullen Demakis and Andrew J. Borst and Florian Praetorius and David Baker},

url = {https://www.nature.com/articles/s41586-024-07813-2, Nature [Open Access]},

doi = {10.1038/s41586-024-07813-2},

year  = {2024},

date = {2024-08-14},

urldate = {2024-08-14},

journal = {Nature},

publisher = {Springer Science and Business Media LLC},

abstract = {Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such ‘action at a distance’ modulation and to create synthetic proteins whose functions can be regulated by effectors. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge. Here, inspired by the classic Monod–Wyman–Changeux model of cooperativity, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules to protein interfaces that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain–side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Peruzzi2024b,

title = {Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions},

author = {Justin A. Peruzzi and Taylor F. Gunnels and Hailey I. Edelstein and Peilong Lu and David Baker and Joshua N. Leonard and Neha P. Kamat},

url = {https://www.nature.com/articles/s41467-024-49678-z [Nature Communications, Open Access]},

doi = {10.1038/s41467-024-49678-z},

year  = {2024},

date = {2024-07-04},

urldate = {2024-12-00},

journal = {Nature Communications},

publisher = {Springer Science and Business Media LLC},

abstract = {Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Yang2024,

title = {Computational design of non-porous pH-responsive antibody nanoparticles},

author = {Erin C. Yang and Robby Divine and Marcos C. Miranda and Andrew J. Borst and Will Sheffler and Jason Z. Zhang and Justin Decarreau and Amijai Saragovi and Mohamad Abedi and Nicolas Goldbach and Maggie Ahlrichs and Craig Dobbins and Alexis Hand and Suna Cheng and Mila Lamb and Paul M. Levine and Sidney Chan and Rebecca Skotheim and Jorge Fallas and George Ueda and Joshua Lubner and Masaharu Somiya and Alena Khmelinskaia and Neil P. King and David Baker},

url = {https://www.nature.com/articles/s41594-024-01288-5, NSMB [Open Access]

https://www.bakerlab.org/wp-content/uploads/2024/05/Yang-etal-NSMB2024-s41594-024-01288-5.pdf, PDF},

doi = {10.1038/s41594-024-01288-5},

year  = {2024},

date = {2024-05-09},

urldate = {2024-05-09},

journal = {Nature Structural & Molecular Biololgy},

publisher = {Springer Science and Business Media LLC},

abstract = {Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and is important for targeted delivery of biologics. Here we describe the design of octahedral non-porous nanoparticles with a targeting antibody on the two-fold symmetry axis, a designed trimer programmed to disassemble below a tunable pH transition point on the three-fold axis, and a designed tetramer on the four-fold symmetry axis. Designed non-covalent interfaces guide cooperative nanoparticle assembly from independently purified components, and a cryo-EM density map closely matches the computational design model. The designed nanoparticles can package protein and nucleic acid payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between 5.9 and 6.7. The ability to incorporate almost any antibody into a non-porous pH-dependent nanoparticle opens up new routes to antibody-directed targeted delivery.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mout2024,

title = {De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity},

author = {Rubul Mout, Ross C. Bretherton, Justin Decarreau, Sangmin Lee, Nicole Gregorio, Natasha I. Edman, Maggie Ahlrichs, Yang Hsia, Danny D. Sahtoe, George Ueda, Alee Sharma, Rebecca Schulman, Cole A. DeForest, David Baker},

url = {https://www.pnas.org/doi/full/10.1073/pnas.2309457121, PNAS [Open Access]},

doi = {10.1073/pnas.2309457121},

year  = {2024},

date = {2024-01-30},

urldate = {2024-01-30},

journal = {Proceedings of the National Academy of Sciences},

abstract = {Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.

},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Goldbach2023,

title = {De novo design of monomeric helical bundles for pH-controlled membrane lysis},

author = {Nicolas Goldbach and Issa Benna and Basile I. M. Wicky and Jacob T. Croft and Lauren Carter and Asim K. Bera and Hannah Nguyen and Alex Kang and Banumathi Sankaran and Erin C. Yang and Kelly K. Lee and David Baker},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.4769, Protein Science

https://www.bakerlab.org/wp-content/uploads/2023/08/Protein-Science-2023-Goldbach.pdf, PDF},

doi = {https://doi.org/10.1002/pro.4769},

year  = {2023},

date = {2023-08-26},

urldate = {2023-08-26},

journal = {Protein Science},

abstract = {Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane lysis tightly restricted to endosomal membranes upon internalization to avoid general membrane insertion and lysis. Here we describe the design of small monomeric proteins with buried histidine containing pH-responsive hydrogen bond networks and membrane permeating amphipathic helices. Of 30 designs that were experimentally tested, all expressed in E. coli, 13 were monomeric with the expected secondary structure, and 4 designs disrupted artificial liposomes in a pH-dependent manner. Mutational analysis showed that the buried histidine hydrogen bond networks mediate pH-responsiveness and control lysis of model membranes within a very narrow range of pH (6.0 - 5.5) with almost no lysis occurring at neutral pH. These tightly controlled lytic monomers could help mediate endosomal escape in designed targeted delivery platforms.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sahtoe2021,

title = {Transferrin receptor targeting by de novo sheet extension},

author = {Sahtoe, Danny D. and Coscia, Adrian and Mustafaoglu, Nur and Miller, Lauren M. and Olal, Daniel and Vulovic, Ivan and Yu, Ta-Yi and Goreshnik, Inna and Lin, Yu-Ru and Clark, Lars and Busch, Florian and Stewart, Lance and Wysocki, Vicki H. and Ingber, Donald E. and Abraham, Jonathan and Baker, David},

url = {https://www.pnas.org/content/118/17/e2021569118, PNAS

},

doi = {10.1073/pnas.2021569118},

year  = {2021},

date = {2021-04-27},

urldate = {2021-04-27},

journal = {Proceedings of the National Academy of Sciences},

abstract = {The de novo design of proteins that bind natural target proteins is useful for a variety of biomedical and biotechnological applications. We describe a design strategy to target proteins containing an exposed beta edge strand. We use the approach to design binders to the human transferrin receptor which shuttles back and forth across the blood{textendash}brain barrier. Such binders could be useful for the delivery of therapeutics into the brain.The de novo design of polar protein{textendash}protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood{textendash}brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.Crystal structures have been deposited in the RCSB PDB with the accession nos. 6WRX, 6WRW, and 6WRV. Additional supporting data has been deposited in the online Zenodo repository (https://zenodo.org/record/4594115) (47). All other study data are included in the article and/or supporting information.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Butterfield2017,

title = {Evolution of a designed protein assembly encapsulating its own RNA genome},

author = {Butterfield, Gabriel L.*

and Lajoie, Marc J.*

and Gustafson, Heather H.

and Sellers, Drew L.

and Nattermann, Una

and Ellis, Daniel

and Bale, Jacob B.

and Ke, Sharon

and Lenz, Garreck H.

and Yehdego, Angelica

and Ravichandran, Rashmi

and Pun, Suzie H.

and King, Neil P.

and Baker, David},

url = {http://dx.doi.org/10.1038/nature25157

https://www.bakerlab.org/wp-content/uploads/2017/12/Nature_Butterfield_et_al_2017.pdf},

doi = {10.1038/nature25157},

issn = {1476-4687},

year  = {2017},

date = {2017-12-13},

journal = {Nature},

abstract = {The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6hours of treatment), and in vivo circulation time (from less than 5minutes to approximately 4.5hours). The resulting synthetic nucleocapsids package one full length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hsia2016,

title = {Design of a hyperstable 60-subunit protein icosahedron},

author = { Yang Hsia* and Jacob B. Bale* and Shane Gonen and Dan Shi and William Sheffler and Kimberly K. Fong and Nattermann and Chunfu Xu and Po-Ssu Huang and Rashmi Ravichandran and Sue Yi and Trisha N. Davis and Tamir Gonen and Neil P. King and David Baker},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/Hsia_Nature_2016.pdf},

doi = {10.1038/nature18010},

year  = {2016},

date = {2016-06-15},

journal = {Nature},

abstract = {The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/ exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{538,

title = {A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells.},

author = { Erik Procko and Geoffrey Y Berguig and Betty W Shen and Yifan Song and Shani Frayo and Anthony J Convertine and Daciana Margineantu and Garrett Booth and Bruno E Correia and Yuanhua Cheng and William R Schief and David M Hockenbery and Oliver W Press and Barry L Stoddard and Patrick S Stayton and David Baker},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Procko2014A.pdf},

doi = {10.1016/j.cell.2014.04.034},

issn = {1097-4172},

year  = {2014},

date = {2014-06-01},

journal = {Cell},

volume = {157},

pages = {1644-56},

abstract = {Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{516,

title = {megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.},

author = { Sandrine Boissel and Jordan Jarjour and Alexander Astrakhan and Andrew Adey and Agn`es Gouble and Philippe Duchateau and Jay Shendure and Barry L Stoddard and Michael T Certo and David Baker and Andrew M Scharenberg},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Boissel_NAR_2013.pdf},

issn = {1362-4962},

year  = {2013},

date = {2013-11-01},

journal = {Nucleic acids research},

abstract = {Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at textquoterightoff-targettextquoteright sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate textquoterightmegaTALtextquoteright, in which the DNA binding region of a transcription activator-like (TAL) effector is used to textquoterightaddresstextquoteright a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{468,

title = {A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly},

author = { Julien R C Bergeron and Liam J Worrall and Nikolaos G Sgourakis and Frank DiMaio and Richard A Pfuetzner and Heather B Felise and Marija Vuckovic and Angel C Yu and Samuel I Miller and David Baker and Natalie C J Strynadka},

url = {http://www.bakerlab.org/wp-content/uploads/2015/12/Bergeron_ppat1003307_13T.pdf},

doi = {10.1371/journal.ppat.1003307},

issn = {1553-7374},

year  = {2013},

date = {2013-04-01},

journal = {PLoS pathogens},

volume = {9},

pages = {e1003307},

abstract = {The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{333,

title = {Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast},

author = { G S Payne and D Baker and E van Tuinen and R Schekman},

url = {https://www.bakerlab.org/wp-content/uploads/2016/06/payne88A.pdf},

issn = {0021-9525},

year  = {1988},

date = {1988-05-01},

journal = {The Journal of cell biology},

volume = {106},

pages = {1453-61},

abstract = {Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}