Journal: PLoS Biology
Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum
Figure Lengend Snippet: Relative affinities of ER-ESCAPE motifs for cargo receptor by analyses of GH steady-state levels. (A) Pre-confluent cells were separately transfected with expression plasmids encoding human GH with 60 different ER-ESCAPE motifs as noted. Eighteen hr posttransfection, cells were washed, extracted with lysis buffer, and analyzed by GH ELISA. GH values (ng GH/mg protein) were normalized within each experiment to the amount of IPV-GH. Each histogram bar represents mean ± SEM. of at least three independent transfections for each construct. (Means of triplicate GH ELISA analyses were used for each extract). Larger error bars among poorest ER-ESCAPE motifs may reflect a variable amount of aggregate formation by larger amounts of accumulated GH. Note that Φ-P-Φ ER-ESCAPE motifs are among the most efficient at trafficking GH, while those including acidic amino acids or glutamines are much less effective. Substitution by positively charged amino acids (generally, R better than K) retained effective trafficking, while loss of proline in position 2 was otherwise detrimental. (B) Prediction of value that each amino acid in positions 1, 2, and 3 adds to the quality of the ER-ESCAPE motif binding to Surf4 based on combination of ELISA and database search results. Note that some combinations may give results not predicted by simply summing a tripeptide’s three individual amino acids contributions indicated by this panel. Φ-P-Φ, hydrophobic-proline-hydrophobic; APV, alanine-proline-valine; DYP, aspartic acid-tyrosine-proline; EEE, glutamic acid–glutamic acid–glutamic acid; EEI, glutamic acid–glutamic acid–isoleucine; EET, glutamic acid–glutamic acid–threonine; EGT, glutamic acid-glycine-threonine; EPA, glutamic acid-proline-alanine; EPT, glutamic acid-proline-threonine; EPV, glutamic acid-proline-valine; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; EST, glutamic acid-serine-threonine; FGT, phenylalanine-glycine-threonine; FLT, phenylalanine-leucine-threonine; FPE, phenylalanine-proline-glutamic acid; FPR, phenylalanine-proline-arginine; FPT, phenylalanine-proline-threonine; FPV, phenylalanine-proline-valine; FQV, phenylalanine-glutamine-valine; FSM, phenylalanine-serine-methionine; FST, phenylalanine-threonine-valine; FTV, phenylalanine-threonine-valine; FVN, phenylalanine-valine-asparagine; GH, growth hormone; GPV, glycine-proline-valine; HSV, histidine-serine-valine; IEV, isoleucine-glycine-valine; IGV, isoleucine-glycine-valine; ILV, isoleucine-leucine-valine; INV, isoleucine-asparagine-valine; IPA, isoleucine-proline-alanine; IPD, isoleucine-proline-aspartic acid; IPE, isoleucine-proline-glutamic acid; IPP, isoleucine-proline-proline; IPS, isoleucine-proline-serine; IPV, isoleucine-proline-valine; IRV, isoleucine-arginine-valine; ISH, isoleucine-serine-histidine; ISP, isoleucine-serine-proline; ISQ, isoleucine-serine-glutamine; ISR, isoleucine-serine-arginine; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; KAV, lysine-alanine-valine; KGV, lysine-glycine-valine; KSV, lysine-serine-valine; KVH, lysine-valine-histidine; NPV, asparagine-proline-valine; QPV, glutamine-proline-valine; QQV, glutamine-glutamine-valine; QSV, glutamine-serine-valine; RGV, arginine-glycine-valine; RLV, arginine-leucine-valine; RPK, arginine-proline-lysine; RPV, arginine-proline-valine; RRR, arginine-arginine-arginine; RSV, arginine-serine-valine; SLT, serine-leucine-threonine; SPT, serine-proline-threonine; SPV, serine-proline-valine; SRT, serine-arginine-threonine; SST, serine-serine-threonine; Surf4, surfeit locus protein 4; YPY, tyrosine-proline-tyrosine.
Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).
Techniques: Transfection, Expressing, Lysis, Enzyme-linked Immunosorbent Assay, Construct, Binding Assay, Indirect Immunoperoxidase Assay, In Situ Hybridization, Single-particle Tracking