When not in functional excess, Surf4 prioritizes ER exit of cargo with stronger ER-ESCAPE motif.

<p>(A) Western blot analyses of cell extracts show steady-state levels of GH starting with strong ER-ESCAPE motif: APV < modest: ITV << nonbinding: EET. Addition of N-linked oligosaccharide (NTT) to fourth amino acid increased the mass/M_r but did not affect relative cellular steady-state levels for any of these proteins, (APV = APV-NTT) < (ITV = ITV-NTT) << (EET = EET-NTT). Coexpression of APV-GH and ITV-GH, irrespective of which construct was glycosylated, did not affect their respective steady-state levels. (B) GH detected in conditioned media (18 hr posttransfection) showed both the remaining complement of APV-GH was trafficked out of cells and the robust nature of HEK293A’s bulk flow process of nonbinding cargo (EET-GH). (C) Partial knockdown of Surf4 with siRNA depleted Surf4 protein levels sufficiently to show GH with strong binding ER-ESCAPE motif (APV-GH) outcompeting modestly binding GH (ITV-GH). HEK293A cells were pretreated for 36 hr with Surf4 siRNA before second transfection with noted GH constructs. Eighteen hr later, washed cells were processed for GH and Surf4 western blots. (D) Quantification of GH western blots shows modestly binding ITV-GH was less effectively trafficked than APV-GH when Surf4 levels were substantially depleted by siRNA. (Error bars are +/− SEM with sample size of <i>n</i> = 4 for inactive siRNA and <i>n</i> = 7 for Surf4 siRNA, <i>P</i> < 0.001.) Three μg cell lysate protein and 6% concentrated media were used for western blot analysis with goat anti-human GH. Six μg cell lysate protein was used for western blot analysis with rabbit anti-Surf4 carboxy-terminal peptide. LI-COR IR-fluorescent second antibodies were used for detection on LI-COR’s Odyssey scanner. Numbers on left are molecular weight standards in kDa. APV, alanine-proline-valine; EET, glutamic acid–glutamic acid–threonine; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; GH, growth hormone; HEK293A, human embryonic kidney cell line 293; IR, infrared; ITV, isoleucine-threonine-valine; NTT, asparagine-threonine-threonine; siRNA, small interfering RNA; Surf4, surfeit locus protein 4.