The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast.
BACKGROUND
In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system. Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin. To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated.


RESULTS
The Saccharomyces cerevisiae SWA2 gene, previously identified in a synthetic lethal screen with arf1, was cloned and found to encode a protein with a carboxy-terminal DnaJ domain which is homologous to that of auxilin. Like auxilin, Swa2p has a clathrin-binding domain and is able to stimulate the ATPase activity of Hsc70. The swa2-1 allele recovered from the original screen carries a point mutation in its tetratricopeptide repeat (TPR) domain, a motif not found in auxilin but known in other proteins to mediate interaction with heat-shock proteins. Swa2p fractionates in the cytosol and appears to be heavily phosphorylated. Disruption of SWA2 causes slow growth and several phenotypes that are very similar to those exhibited by clathrin mutants. Furthermore, the swa2Delta mutant exhibits a significant increase in membrane- associated or -assembled clathrin relative to a wild-type strain.


CONCLUSIONS
These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo. They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.
Gall
W E
WE
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.
Higginbotham
M A
MA
Chen
C
C
Ingram
M F
MF
Cyr
D M
DM
Graham
T R
TR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D002966:Clathrin; D018840:HSP70 Heat-Shock Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C419865:SWA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins