Proteins and lipids of the plasma membrane underlie constant remodeling via a combination of the secretory- and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted for recycling to the plasma membrane or degradation in vacuoles. Previously we have shown a role for the GARP complex in sphingolipid sorting and homeostasis (Fröhlich et al. 2015). However, the majority of cargo sorted in a GARP dependent process remain largely unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry based vacuolar proteomics and lipidomics to show that recycling of two specific groups of proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of the lipid composition are the first occurring defects in GARP mutants. Our assay can be adapted to systematically map cargo of the entire endocytic pathway.

30Proteins and lipids of the plasma membrane underlie constant remodeling via a combination 31 of the secretory-and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted 32 for recycling to the plasma membrane or degradation in vacuoles. In a previous paper we have 33 shown a role for the GARP complex in sphingolipid sorting and homeostasis (Fröhlich et al. 34 2015). However, the majority of cargo sorted in a GARP dependent process remain largely 35 unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry 36 based vacuolar proteomics and lipidomics to show that recycling of two specific groups of 37 proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a 38 functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of 39 the lipid composition are the first occurring defects in GARP mutants. Our assay can be 40 adapted to systematically map cargo of the entire endocytic pathway. al., 2010). Deletion of the GARP complex has been linked to multiple cellular dysfunctions.94 The first discovered and canonical pathway is the sorting of the carboxy peptidase Y (CPY) 95 receptor Vps10 (Conibear and Stevens, 2000), hence the name Vps of all subunits. However, 96 deletion of the GARP complex has also been linked to defects in autophagy and mitochondrial 97 tubulation (Reggiori and Klionsky, 2006), defects in the actin cytoskeleton (Fiedler et al., 2002), 98 4 cell wall integrity (Conde et al., 2003), vacuole integrity (Conibear and Stevens, 2000) and 99 several more (for a complete overview see ; Bonifacino and Hierro, 2011). 100We have previously identified an important role for the GARP complex in lipid homeostasis 101 (Fröhlich et al., 2015). Deletion of either subunit of the GARP complex results in the massive 102 accumulation of sphingolipid intermediates, the long chain bases (LCBs) in cells. Interestingly 103 all observed defects in GARP knockout mutants, including vacuolar fragmentation can be 104 rescued by chemical depletion of sphingolipids. This suggests that sphingolipid accumulation 105 is the causing problem in cells but the molecular mechanism for this remains largely elusive. 106Here, we have developed a system that combines auxin induced degradation of the GARP 107 complex with mass spectrometry based vacuolar proteomics and lipidomics to systematically 108 identify cargo of the GARP dependent endosomal sorting pathway. We show that plasma 109 membrane proteins of two different functional groups, amino-phospholipid flippases and cell 110 wall biosynthesis proteins, are the first to be mis-sorted after chemical depletion of the GARP 111 complex. We also analyze the cellular and vacuolar lipid composition to shed some light on 112 the important functions of the GARP complex in cells.

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