Cargo selectivity of yeast sorting nexins

Abstract

Sorting nexins are PX domain-containing proteins that bind phospholipids and often act in membrane trafficking where they help to select cargo. However, the functions and cargo specificities of many sorting nexins are unknown. Here, a high-throughput imaging screen was used to identify new sorting nexin cargo in the yeast Saccharomyces cerevisiae. Deletions of 9 different sorting nexins were screened for mislocalization of a set of green fluorescent protein (GFP)-tagged membrane proteins found at the plasma membrane, Golgi or endosomes. This identified 27 proteins that require 1 or more sorting nexins for their correct localization, 23 of which represent novel sorting nexin cargo. Nine hits whose sorting was dependent on Snx4, the sorting nexin-containing retromer complex, or both retromer and Snx3, were examined in detail to search for potential sorting motifs. We identified cytosolic domains of Ear1, Ymd8 and Ymr010w that conferred retromer-dependent sorting on a chimeric reporter and identified conserved residues required for this sorting in a functional assay. This work defined a consensus sequence for retromer and Snx3-dependent sorting. K E Y W O R D S

Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes

“…In yeast, these include the iron transporter Ftr1, the SNARE protein Pep12, and two membrane-bound proteases, Ste13 and Kex2 (Voos and Stevens, 1998; Hettema et al , 2003; Strochlic et al , 2007), whereas the Wnt receptor Wntless and the transferrin receptor are sorted by Snx3 in metazoans (Harterink et al , 2011; Chen et al , 2013). The short conserved sorting motif that we identified in the cytoplasmic N-terminal tail of Neo1 contains a core FEM tripeptide that has some similarity to signals in Ste13 and Kex2 (Wilcox et al , 1992; Nothwehr et al , 1993; Voos and Stevens, 1998) and fits a recently defined Y/F-x-Φ consensus (where Φ is a bulky hydrophobic residue) implicated in retromer and Snx3-based sorting in a large-scale study (Bean et al , 2017). This tripeptide is frequently associated with conserved acidic and hydrophobic residues.…”

The chaperone Chs7 forms a stable complex with Chs3 and promotes its activity at the cell surface

“…We used strains carrying mutations in different transport steps to examine the trafficking itineraries of Chs3 and variant forms of Chs7. Chs3 has sorting signals for binding AP‐1, retromer and exomer, and is retained intracellularly in exomer mutants by continuous AP‐1‐dependent cycling between Golgi and endosomes . In chs6∆ mutants, Chs7‐GFP was trapped in intracellular Golgi/endosomal compartments, but was sorted to the vacuole when the Chs3 interaction was abrogated by mutation of the Chs7 tail (Figure B).…”

Autophagic clearance of proteasomes in yeast requires the conserved sorting nexin Snx4

“…proteasomes into phase-separated granules similar to those observed in response to carbon deprivation (62) or is instead involved in the enrichment of proteasomes in membrane-encased structures that are subsequently undergoing Atg8-dependent engulfment and delivery to the vacuole. Considering that Snx4 is a PI3P-binding protein involved in membrane protein sorting (37,40,41) and that its membrane-binding function is necessary for proteasome autophagy (Fig. 7), we favor the latter possibility.…”

“…SNX4 family nexins typically function as dimers. In budding yeast, Snx4 can heterodimerize with two other PX-BAR family nexins, Snx41 and Snx42, to form Snx4-Snx41 and Snx4-Snx42 complexes with distinct and overlapping functions (37,40,41,43). In S. cerevisiae, Snx4 cooperates with Snx41 to promote retrograde sorting of the autophagy integral membrane protein Atg27, whereas Snx42 is dispensable (37).…”