Molecular dissection of Erv26p identifies separable cargo binding and coat protein sorting activities.
Efficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport.
Bue
Catherine A
CA
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Barlowe
Charles
C
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D022181:COP-Coated Vesicles / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
C503875:SVP26 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D008364:Mannosyltransferases; D000469:Alkaline Phosphatase