Adaptor complex-independent clathrin function in yeast.

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

[1]  D. Branton,et al.  Assembly units of clathrin coats , 1981, Nature.

[2]  J. Bonifacino,et al.  Association of the AP-3 adaptor complex with clathrin. , 1998, Science.

[3]  J. Carbon,et al.  Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. , 1980, Gene.

[4]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[5]  K. Nathanson,et al.  Structural and functional division into two domains of the large (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[7]  S. Harrison,et al.  Functional organization of clathrin in coats: combining electron cryomicroscopy and X-ray crystallography. , 1999, Molecular cell.

[8]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[9]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[10]  J. Rizo,et al.  The LDL Receptor Clustering Motif Interacts with the Clathrin Terminal Domain in a Reverse Turn Conformation , 1998, The Journal of cell biology.

[11]  H. Riezman,et al.  Clathrin functions in the absence of heterotetrameric adaptors and AP180‐related proteins in yeast , 1999, The EMBO journal.

[12]  J. Dixon,et al.  Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. , 1991, Analytical biochemistry.

[13]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[14]  J. Keen,et al.  Clathrin assembly proteins: affinity purification and a model for coat assembly , 1987, The Journal of cell biology.

[15]  J. Bonifacino,et al.  Interaction of tyrosine-based sorting signals with clathrin-associated proteins. , 1995, Science.

[16]  R. Crowther,et al.  Location of the 100 kd‐50 kd accessory proteins in clathrin coats. , 1986, The EMBO journal.

[17]  N Grigorieff,et al.  Clathrin coats at 21 Å resolution: a cellular assembly designed to recycle multiple membrane receptors , 1998, The EMBO journal.

[18]  Scott D Emr,et al.  The AP-3 Adaptor Complex Is Essential for Cargo-Selective Transport to the Yeast Vacuole , 1997, Cell.

[19]  Richard G. W. Anderson,et al.  The Appendage Domain of α-Adaptin Is a High Affinity Binding Site for Dynamin (*) , 1995, The Journal of Biological Chemistry.

[20]  J. Thorner,et al.  Enzymes required for yeast prohormone processing. , 1988, Annual review of physiology.

[21]  G. Payne,et al.  The Saccharomyces cerevisiae APS1 gene encodes a homolog of the small subunit of the mammalian clathrin AP‐1 complex: evidence for functional interaction with clathrin at the Golgi complex. , 1994, The EMBO journal.

[22]  M. Robinson,et al.  Purification and properties of 100‐kd proteins from coated vesicles and their reconstitution with clathrin. , 1984, The EMBO journal.

[23]  S. Schmid,et al.  Clathrin-coated vesicle formation and protein sorting: an integrated process. , 1997, Annual review of biochemistry.

[24]  G. Sprague,,et al.  Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors , 1993, The Journal of cell biology.

[25]  J. Bonifacino,et al.  AP-4, a Novel Protein Complex Related to Clathrin Adaptors* , 1999, The Journal of Biological Chemistry.

[26]  S. Emr,et al.  Yeast epsins contain an essential N‐terminal ENTH domain, bind clathrin and are required for endocytosis , 1999, The EMBO journal.

[27]  P. Evans,et al.  A Structural Explanation for the Binding of Multiple Ligands by the α-Adaptin Appendage Domain , 1999, Cell.

[28]  G. Bush,et al.  Secretion in yeast. Purification and in vitro translocation of chemical amounts of prepro-alpha-factor. , 1991, Journal of Biological Chemistry.

[29]  Diana S Chu,et al.  The Light Chain Subunit Is Required for Clathrin Function in Saccharomyces cerevisiae* , 1996, The Journal of Biological Chemistry.

[30]  Scott D. Emr,et al.  Pan1p, Yeast eps15, Functions as a Multivalent Adaptor That Coordinates Protein–Protein Interactions Essential for Endocytosis , 1998, The Journal of cell biology.

[31]  J. Bonifacino,et al.  Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor. , 1999, Molecular cell.

[32]  S. Emr,et al.  A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast , 1995, The Journal of cell biology.

[33]  R. Deschenes,et al.  Vectors for the inducible overexpression of glutathione S‐transferase fusion proteins in yeast , 1993, Yeast.

[34]  K. von Figura,et al.  Early Embryonic Death of Mice Deficient in γ-Adaptin* , 1999, The Journal of Biological Chemistry.

[35]  P. De Camilli,et al.  A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Silverman.  Methods in yeast genetics (laboratory course manual): By F. Sherman, G. R. Fink, and J. B. Hicks, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1987. 186 pp. Paper bound, $25.00 , 1987 .

[37]  S. Lemmon,et al.  The Yeast Adaptor Protein Complex, AP-3, Is Essential for the Efficient Delivery of Alkaline Phosphatase by the Alternate Pathway to the Vacuole , 1997, The Journal of cell biology.

[38]  T. Kirchhausen,et al.  Dileucine‐based sorting signals bind to the β chain of AP‐1 at a site distinct and regulated differently from the tyrosine‐based motif‐binding site , 1998, The EMBO journal.

[39]  S. Hunt,et al.  Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis. , 1997, Molecular biology of the cell.

[40]  P. Evans,et al.  A structural explanation for the binding of multiple ligands by the alpha-adaptin appendage domain. , 1999, Cell.

[41]  H. McMahon.  Endocytosis: An assembly protein for clathrin cages , 1999, Current Biology.

[42]  G. Payne,et al.  A dileucine‐like sorting signal directs transport into an AP‐3‐dependent, clathrin‐independent pathway to the yeast vacuole , 1998, The EMBO journal.

[43]  G. Sprague,,et al.  Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast , 1993, The Journal of cell biology.

[44]  G. Payne,et al.  A modulatory role for clathrin light chain phosphorylation in Golgi membrane protein localization during vegetative growth and during the mating response of Saccharomyces cerevisiae. , 1999, Molecular biology of the cell.

[45]  G. Payne,et al.  Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae , 1992, The Journal of cell biology.

[46]  W. B. Snyder,et al.  Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component , 1997, The EMBO journal.

[47]  S. Emr,et al.  The AP-3 complex: a coat of many colours. , 1998, Trends in cell biology.

[48]  R. Rothstein.  Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. , 1991, Methods in enzymology.

[49]  W. N. Burnette,et al.  "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. , 1981, Analytical biochemistry.

[50]  T. Stevens,et al.  The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway , 1997, The Journal of cell biology.

[51]  R A Crowther,et al.  Three‐dimensional structure of clathrin cages in ice. , 1986, The EMBO journal.

[52]  L. C. Robinson,et al.  Suppressors of YCK‐encoded yeast casein kinase 1 deficiency define the four subunits of a novel clathrin AP‐like complex , 1997, The EMBO journal.

[53]  D. Botstein,et al.  Structure and function of the yeast URA3 gene: expression in Escherichia coli. , 1984, Gene.

[54]  R. Darnell,et al.  A novel adaptor-related protein complex , 1996, The Journal of cell biology.

[55]  A. Dautry‐Varsat,et al.  The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2 , 1995, The Journal of cell biology.

[56]  M. Robinson,et al.  Characterization of the Adaptor-related Protein Complex, AP-3 , 1997, The Journal of cell biology.

[57]  J. Hargreaves,et al.  A gene encoding γ-adaptin is required for apical extension growth in Ustilago maydis , 1995 .

[58]  T. Stevens,et al.  Vacuole Biogenesis in Saccharomyces cerevisiae: Protein Transport Pathways to the Yeast Vacuole , 1998, Microbiology and Molecular Biology Reviews.

[59]  T. Kirchhausen,et al.  A late Golgi sorting function for Saccharomyces cerevisiae Apm1p, but not for Apm2p, a second yeast clathrin AP medium chain-related protein. , 1995, Molecular biology of the cell.

[60]  J. Benovic,et al.  Arrestin/Clathrin Interaction , 1997, The Journal of Biological Chemistry.

[61]  K. Blumer,et al.  Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast. , 1997, Journal of cell science.

[62]  H. Jäckle,et al.  Role of Drosophila alpha-adaptin in presynaptic vesicle recycling. , 1997, Cell.

[63]  M. Robinson,et al.  Clathrin and adaptors. , 1998, Biochimica et biophysica acta.

[64]  B. Hoflack,et al.  Mechanisms of protein sorting and coat assembly: insights from the clathrin-coated vesicle pathway. , 1998, Current opinion in cell biology.

[65]  J. Benovic,et al.  Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. , 1996, Nature.

[66]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[67]  S. Harrison,et al.  Protein organization in clathrin trimers , 1981, Cell.

[68]  H. McMahon,et al.  Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain , 1997, Current Biology.

[69]  G. Payne,et al.  Clathrin Coats— Threads Laid Bare , 1998, Cell.

[70]  R. Schekman,et al.  Clathrin: a role in the intracellular retention of a Golgi membrane protein. , 1989, Science.

[71]  A. Myers,et al.  Yeast/E. coli shuttle vectors with multiple unique restriction sites , 1986, Yeast.

[72]  E. Ungewickell,et al.  Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. , 1988, The EMBO journal.

[73]  Pier Paolo Di Fiore,et al.  Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis , 1998, Nature.

[74]  T. Kirchhausen,et al.  A Clathrin-binding Site in the Hinge of the 2 Chain of Mammalian AP-2 Complexes (*) , 1995, The Journal of Biological Chemistry.

[75]  R. Schekman,et al.  Coat Proteins and Vesicle Budding , 1996, Science.

[76]  G. Payne,et al.  A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. , 1992, The EMBO journal.

[77]  D. Klionsky.  Nonclassical Protein Sorting to the Yeast Vacuole* , 1998, The Journal of Biological Chemistry.

[78]  T. Kirchhausen,et al.  The beta 1 and beta 2 subunits of the AP complexes are the clathrin coat assembly components. , 1993, The EMBO journal.

[79]  P. Sternberg,et al.  unc-101, a gene required for many aspects of Caenorhabditis elegans development and behavior, encodes a clathrin-associated protein. , 1994, Genes & Development.

[80]  M. Robinson.  100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies , 1987, The Journal of cell biology.