Identification of the yeast R-SNARE Nyv1p as a novel longin domain-containing protein.
Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXX phi-like adaptin-dependent sorting signal (Y31GTI34) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y31GTI34 (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y31GTI34 motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXX phi-like sorting signals.
Wen
Wenyu
W
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Special Administrative Region of China.
Chen
Lu
L
Wu
Hao
H
Sun
Xin
X
Zhang
Mingjie
M
Banfield
David K
DK
D033963:Adaptor Protein Complex 3 / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001692:Biological Transport; D049452:Green Fluorescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D033963:Adaptor Protein Complex 3; D008565:Membrane Proteins; C500806:Nyv1 protein, S cerevisiae; D050683:R-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins