Efficient ER Exit and Vacuole Targeting of Yeast Sna2p Require Two Tyrosine‐Based Sorting Motifs

SNA (Sensitive to Na+) proteins form a membrane protein family, which, in the yeast Saccharomyces cerevisiae, is composed of four members: Sna1p/Pmp3p, Sna2p, Sna3p and Sna4p. In this study, we focused on the 79 residue Sna2p protein. We found that Sna2p is localized in the vacuolar membrane. Directed mutagenesis showed that two functional tyrosine motifs YXXØ are present in the C‐terminal region. Each of these is involved in a different Golgi‐to‐vacuole targeting pathway: the tyrosine 65 motif is involved in adaptor protein (AP‐1)‐dependent targeting, whereas the tyrosine 75 motif is involved in AP‐3‐dependent targeting. Moreover, our data suggest that these motifs also play a crucial role in the exit of Sna2p from the endoplasmic reticulum (ER). Directed mutagenesis of these tyrosines led to a partial redirection of Sna2p to lipid bodies, probably because of a decrease in ER exit efficiency. Sna2p is the first yeast protein in which two YXXØ motifs have been identified and both were shown to be functional at two different steps of the secretory pathway, ER exit and Golgi‐to‐vacuole transport.

[1]  E. Leitner,et al.  Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae. , 2009, Biochimica et biophysica acta.

[2]  W. Pokrzywa,et al.  Dual Sorting of the Saccharomyces cerevisiae Vacuolar Protein Sna4p , 2009, Eukaryotic Cell.

[3]  C. Thiele,et al.  Cell biology of lipid droplets. , 2008, Current opinion in cell biology.

[4]  R. Haguenauer‐Tsapis,et al.  Targeting of Sna3p to the Endosomal Pathway Depends on Its Interaction with Rsp5p and Multivesicular Body Sorting on Its Ubiquitylation , 2007, Traffic.

[5]  A. Nakano,et al.  Mechanisms of COPII vesicle formation and protein sorting , 2007, FEBS letters.

[6]  K. Athenstaedt,et al.  Synthesis, storage and degradation of neutral lipids in yeast. , 2007, Biochimica et biophysica acta.

[7]  P. Piper,et al.  Fab1p and AP-1 are required for trafficking of endogenously ubiquitylated cargoes to the vacuole lumen in S. cerevisiae , 2006, Journal of Cell Science.

[8]  Mingjie Zhang,et al.  Identification of the yeast R-SNARE Nyv1p as a novel longin domain-containing protein. , 2006, Molecular biology of the cell.

[9]  Yingming Zhao,et al.  An intimate collaboration between peroxisomes and lipid bodies , 2006, The Journal of cell biology.

[10]  T. Stevens,et al.  Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae. , 2005, Biochimica et biophysica acta.

[11]  T. Heineman,et al.  Conserved cytoplasmic domain sequences mediate the ER export of VZV, HSV-1, and HCMV gB. , 2004, Virology.

[12]  M. Moloney,et al.  Membrane topology and sequence requirements for oil body targeting of oleosin. , 2004, The Plant journal : for cell and molecular biology.

[13]  J. Bonifacino,et al.  The Mechanisms of Vesicle Budding and Fusion , 2004, Cell.

[14]  A. Ostermeyer,et al.  Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets , 2004, The Journal of cell biology.

[15]  Linyi Chen,et al.  The yeast casein kinase Yck3p is palmitoylated, then sorted to the vacuolar membrane with AP-3-dependent recognition of a YXXPhi adaptin sorting signal. , 2003, Molecular biology of the cell.

[16]  J. Bonifacino,et al.  Signals for sorting of transmembrane proteins to endosomes and lysosomes. , 2003, Annual review of biochemistry.

[17]  E. O’Shea,et al.  Global analysis of protein localization in budding yeast , 2003, Nature.

[18]  P. De Camilli,et al.  The synaptojanin-like protein Inp53/Sjl3 functions with clathrin in a yeast TGN-to-endosome pathway distinct from the GGA protein-dependent pathway. , 2003, Molecular biology of the cell.

[19]  R. Schekman,et al.  Concentrative sorting of secretory cargo proteins into COPII-coated vesicles , 2002, The Journal of cell biology.

[20]  C. Barlowe,et al.  The Erv41p–Erv46p complex: multiple export signals are required in trans for COPII‐dependent transport from the ER , 2002, The EMBO journal.

[21]  F. Cordes,et al.  Proline-induced distortions of transmembrane helices. , 2002, Journal of molecular biology.

[22]  E. Epping,et al.  Identification of Interdependent Signals Required for Anterograde Traffic of the ATP-binding Cassette Transporter Protein Yor1p* , 2002, The Journal of Biological Chemistry.

[23]  A. Nakano,et al.  Emp47p and its close homolog Emp46p have a tyrosine-containing endoplasmic reticulum exit signal and function in glycoprotein secretion in Saccharomyces cerevisiae. , 2002, Molecular biology of the cell.

[24]  R. Schekman,et al.  The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins. , 2002, Developmental cell.

[25]  S. Stymne,et al.  Storage Lipid Synthesis Is Non-essential in Yeast* , 2002, The Journal of Biological Chemistry.

[26]  J. McLauchlan,et al.  The Domains Required to Direct Core Proteins of Hepatitis C Virus and GB Virus-B to Lipid Droplets Share Common Features with Plant Oleosin Proteins* , 2002, The Journal of Biological Chemistry.

[27]  R. Jirtle.  Mannose 6-Phosphate/Insulin-Like Growth Factor II Receptor , 2002 .

[28]  D. Gallwitz,et al.  An acidic sequence of a putative yeast Golgi membrane protein binds COPII and facilitates ER export , 2001, The EMBO journal.

[29]  J. Tzen,et al.  An in vitro system to examine the effective phospholipids and structural domain for protein targeting to seed oil bodies. , 2001, Plant & cell physiology.

[30]  Fulvio Reggiori,et al.  Sorting of proteins into multivesicular bodies: ubiquitin‐dependent and ‐independent targeting , 2001, The EMBO journal.

[31]  J. Bonifacino,et al.  Adaptor-related proteins. , 2001, Current opinion in cell biology.

[32]  G. Payne,et al.  Clathrin Interactions with C‐Terminal Regions of the Yeast AP‐1 β and γ Subunits are Important for AP‐1 Association with Clathrin Coats , 2001, Traffic.

[33]  S. Munro,et al.  Accumulation of Caveolin in the Endoplasmic Reticulum Redirects the Protein to Lipid Storage Droplets , 2001, The Journal of cell biology.

[34]  J. Lippincott-Schwartz,et al.  The Sar1 Gtpase Coordinates Biosynthetic Cargo Selection with Endoplasmic Reticulum Export Site Assembly , 2001, The Journal of cell biology.

[35]  K. Athenstaedt,et al.  Intracellular lipid particles of eukaryotic cells. , 2000, Biochimica et biophysica acta.

[36]  A. Goffeau,et al.  Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane , 2000, The EMBO journal.

[37]  G. Payne,et al.  Adaptor complex-independent clathrin function in yeast. , 1999, Molecular biology of the cell.

[38]  S. Kohlwein,et al.  Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae , 1999, Journal of bacteriology.

[39]  W. Balch,et al.  A Di-acidic (DXE) Code Directs Concentration of Cargo during Export from the Endoplasmic Reticulum* , 1999, The Journal of Biological Chemistry.

[40]  S. Emr,et al.  The AP-3 complex: a coat of many colours. , 1998, Trends in cell biology.

[41]  C. Burd,et al.  Acidic Di-leucine Motif Essential for AP-3–dependent Sorting and Restriction of the Functional Specificity of the Vam3p Vacuolar t-SNARE , 1998, The Journal of cell biology.

[42]  G. Payne,et al.  A dileucine‐like sorting signal directs transport into an AP‐3‐dependent, clathrin‐independent pathway to the yeast vacuole , 1998, The EMBO journal.

[43]  Scott D Emr,et al.  The AP-3 Adaptor Complex Is Essential for Cargo-Selective Transport to the Yeast Vacuole , 1997, Cell.

[44]  L. C. Robinson,et al.  Suppressors of YCK‐encoded yeast casein kinase 1 deficiency define the four subunits of a novel clathrin AP‐like complex , 1997, The EMBO journal.

[45]  G. Daum,et al.  Characterization of lipid particles of the yeast, Saccharomyces cerevisiae , 1994, Yeast.

[46]  G. Payne,et al.  The Saccharomyces cerevisiae APS1 gene encodes a homolog of the small subunit of the mammalian clathrin AP‐1 complex: evidence for functional interaction with clathrin at the Golgi complex. , 1994, The EMBO journal.

[47]  S. Kornfeld,et al.  Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor. , 1992, The Journal of biological chemistry.

[48]  R. Schiestl,et al.  Improved method for high efficiency transformation of intact yeast cells. , 1992, Nucleic acids research.

[49]  S. Kornfeld,et al.  Localization of the signal for rapid internalization of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail. , 1991, The Journal of biological chemistry.

[50]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[51]  C. Sevier,et al.  Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs. , 2000, Molecular biology of the cell.

[52]  T. Kirchhausen,et al.  A late Golgi sorting function for Saccharomyces cerevisiae Apm1p, but not for Apm2p, a second yeast clathrin AP medium chain-related protein. , 1995, Molecular biology of the cell.

[53]  R. Müller,et al.  Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. , 1994, Nucleic acids research.