A large family of endosome-localized proteins related to sorting nexin 1

2001 ◽  
Vol 358 (1) ◽  
pp. 7-16 ◽  
Author(s):  
Rohan D. TEASDALE ◽  
David LOCI ◽  
Fiona HOUGHTON ◽  
Lars KARLSSON ◽  
Paul A. GLEESON
Keyword(s):  
Fluorescent Protein ◽  
Expressed Sequence Tag ◽  
Intracellular Localization ◽  
Coiled Coil ◽  
Large Family ◽  
Early Endosome ◽  
Cell Surface Expression ◽  
Sorting Nexin ◽  
Px Domain ◽  
Sorting Nexin 1

Sorting nexin 1 (SNX1), a peripheral membrane protein, has previously been shown to regulate the cell-surface expression of the human epidermal growth factor receptor [Kurten, Cadena and Gill (1996) Science 272, 1008–1010]. Searches of human expressed sequence tag databases with SNX1 revealed eleven related human cDNA sequences, termed SNX2 to SNX12, eight of them novel. Analysis of SNX1-related sequences in the Saccharomyces cerevisiae genome clearly shows a greatly expanded SNX family in humans in comparison with yeast. On the basis of the predicted protein sequences, all members of this family of hydrophilic molecules contain a conserved 70–110-residue Phox homology (PX) domain, referred to as the SNX-PX domain. Within the SNX family, subgroups were identified on the basis of the sequence similarities of the SNX-PX domain and the overall domain structure of each protein. The members of one subgroup, which includes human SNX1, SNX2, SNX4, SNX5 and SNX6 and the yeast Vps5p and YJL036W, all contain coiled-coil regions within their large C-terminal domains and are found distributed in both membrane and cytosolic fractions, typical of hydrophilic peripheral membrane proteins. Localization of the human SNX1 subgroup members in HeLa cells transfected with the full-length cDNA species revealed a similar intracellular distribution that in all cases overlapped substantially with the early endosome marker, early endosome autoantigen 1. The intracellular localization of deletion mutants and fusions with green fluorescent protein showed that the C-terminal regions of SNX1 and SNX5 are responsible for their endosomal localization. On the basis of these results, the functions of these SNX molecules are likely to be unique to endosomes, mediated in part by interactions with SNX-specific C-terminal sequences and membrane-associated determinants.

scholarly journals Determinants of the Endosomal Localization of Sorting Nexin 1

2005 ◽  
Vol 16 (4) ◽  
pp. 2049-2057 ◽  
Author(s):  
Qi Zhong ◽  
Martin J. Watson ◽  
Cheri S. Lazar ◽  
Andrea M. Hounslow ◽  
Jonathan P. Waltho ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Early Endosome ◽  
Nmr Structure ◽  
Sorting Nexin ◽  
High Affinity ◽  
Terminal Domain ◽  
Px Domain ◽  
Phox Homology ◽  
Phosphatidylinositol 3

The sorting nexin (SNX) family of proteins is characterized by sequence-related phox homology (PX) domains. A minority of PX domains bind with high affinity to phosphatidylinositol 3-phosphate [PI(3)P], whereas the majority of PX domains exhibit low affinity that is insufficient to target them to vesicles. SNX1 is located on endosomes, but its low affinity PX domain fails to localize in vivo. The NMR structure of the PX domain of SNX1 reveals an overall fold that is similar to high-affinity PX domains. However, the phosphatidylinositol (PI) binding pocket of the SNX1 PX domain is incomplete; regions of the pocket that are well defined in high-affinity PX domains are highly mobile in SNX1. Some of this mobility is lost upon binding PI(3)P. The C-terminal domain of SNX1 is a long helical dimer that localizes to vesicles but not to the early endosome antigen-1–containing vesicles where endogenous SNX1 resides. Thus, the obligate dimerization of SNX1 that is driven by the C-terminal domain creates a high-affinity PI binding species that properly targets the holo protein to endosomes.

scholarly journals A large family of endosome-localized proteins related to sorting nexin 1

2001 ◽  
Vol 358 (1) ◽  
pp. 7 ◽  
Author(s):  
Rohan D. TEASDALE ◽  
David LOCI ◽  
Fiona HOUGHTON ◽  
Lars KARLSSON ◽  
Paul A. GLEESON
Keyword(s):  
Large Family ◽  
Sorting Nexin ◽  
Sorting Nexin 1

scholarly journals Luv1p/Rki1p/Tcs3p/Vps54p, a Yeast Protein That Localizes to the Late Golgi and Early Endosome, Is Required for Normal Vacuolar Morphology.

2000 ◽  
Vol 11 (7) ◽  
pp. 2429-2443 ◽  
Author(s):  
Michael J. Conboy ◽  
Martha S. Cyert
Keyword(s):  
Fluorescent Protein ◽  
Protein A ◽  
Coiled Coil ◽  
Specific Inhibitor ◽  
Early Endosome ◽  
Carboxypeptidase Y ◽  
Similar Drug ◽  
Green Fluorescent ◽  
Vacuolar Protein ◽  
Golgi Network

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. Theluv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth.luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H+-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn2+, Mn2+, and Cd2+) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 × g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein–Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.

Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

2022 ◽  
Author(s):  
Maciek Adamowski ◽  
Ivana Matijević ◽  
Jiří Friml
Keyword(s):  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Large Family ◽  
Adaptor Proteins ◽  
Serine Phosphorylation ◽  
Binding Motif ◽  
Double Knockout ◽  
Coated Pits ◽  
Knockout Mutants ◽  
Golgi Network

Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.

Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide

2010 ◽  
Vol 299 (2) ◽  
pp. G320-G328 ◽  
Author(s):  
Claudia Stross ◽  
Angelika Helmer ◽  
Katrin Weissenberger ◽  
Boris Görg ◽  
Verena Keitel ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bile Salt ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Sodium Taurocholate ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Salt Uptake ◽  
Kinase C

Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca2+-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na+-K+-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 ± 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca2+-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.

scholarly journals A Role for Sorting Nexin 2 in Epidermal Growth Factor Receptor Down-regulation: Evidence for Distinct Functions of Sorting Nexin 1 and 2 in Protein Trafficking

2004 ◽  
Vol 15 (5) ◽  
pp. 2143-2155 ◽  
Author(s):  
Anuradha Gullapalli ◽  
Tiana A. Garrett ◽  
May M. Paing ◽  
Courtney T. Griffin ◽  
Yonghua Yang ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Protein Trafficking ◽  
Growth Factor Receptor ◽  
Down Regulation ◽  
Sorting Nexin ◽  
Px Domain ◽  
Early Endosomes ◽  
Epidermal Growth

Sorting nexin 1 (SNX1) and SNX2, homologues of the yeast vacuolar protein-sorting (Vps)5p, contain a phospholipid-binding motif termed the phox homology (PX) domain and a carboxyl terminal coiled-coil region. A role for SNX1 in trafficking of cell surface receptors from endosomes to lysosomes has been proposed; however, the function of SNX2 remains unknown. Toward understanding the function of SNX2, we first examined the distribution of endogenous protein in HeLa cells. We show that SNX2 resides primarily in early endosomes, whereas SNX1 is found partially in early endosomes and in tubulovesicular-like structures distributed throughout the cytoplasm. We also demonstrate that SNX1 interacts with the mammalian retromer complex through its amino terminal domain, whereas SNX2 does not. Moreover, activated endogenous epidermal growth factor receptor (EGFR) colocalizes markedly with SNX2-positive endosomes, but minimally with SNX1-containing vesicles. To assess SNX2 function, we examined the effect of a PX domain-mutated SNX2 that is defective in vesicle localization on EGFR trafficking. Mutant SNX2 markedly inhibited agonist-induced EGFR degradation, whereas internalization remained intact. In contrast, SNX1 PX domain mutants failed to effect EGFR degradation, whereas a SNX1 deletion mutant significantly inhibited receptor down-regulation. Interestingly, knockdown of SNX1 and SNX2 expression by RNA interference failed to alter agonist-induced EGFR down-regulation. Together, these findings suggest that both SNX1 and SNX2 are involved in regulating lysosomal sorting of internalized EGFR, but neither protein is essential for this process. These studies are the first to demonstrate a function for SNX2 in protein trafficking.

scholarly journals Hydrophobic Regions Adjacent to Transmembrane Domains 1 and 5 Are Important for the Targeting of the 70-kDa Peroxisomal Membrane Protein

2007 ◽  
Vol 282 (46) ◽  
pp. 33831-33844 ◽  
Author(s):  
Yoshinori Kashiwayama ◽  
Kota Asahina ◽  
Masashi Morita ◽  
Tsuneo Imanaka
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein

The 70-kDa peroxisomal membrane protein (PMP70) is a major component of peroxisomal membranes. Human PMP70 consists of 659 amino acid residues and has six putative transmembrane domains (TMDs). PMP70 is synthesized on cytoplasmic ribosomes and targeted posttranslationally to peroxisomes by an unidentified peroxisomal membrane protein targeting signal (mPTS). In this study, to examine the mPTS within PMP70 precisely, we expressed various COOH-terminally or NH2-terminally deleted constructs of PMP70 fused with green fluorescent protein (GFP) in Chinese hamster ovary cells and determined their intracellular localization by immunofluorescence. In the COOH-terminally truncated PMP70, PMP70(AA.1-144)-GFP, including TMD1 and TMD2 of PMP70, was still localized to peroxisomes. However, by further removal of TMD2, PMP70(AA.1-124)-GFP lost the targeting ability, and PMP70(TMD2)-GFP did not target to peroxisomes by itself. The substitution of TMD2 in PMP70(AA.1-144)-GFP for TMD4 or TMD6 did not affect the peroxisomal localization, suggesting that PMP70(AA.1-124) contains the mPTS and an additional TMD is required for the insertion into the peroxisomal membrane. In the NH2-terminal 124-amino acid region, PMP70 possesses hydrophobic segments in the region adjacent to TMD1. By the disruption of these hydrophobic motifs by the mutation of L21Q/L22Q/L23Q or I70N/L71Q, PMP70(AA.1-144)-GFP lost targeting efficiency. The NH2-terminally truncated PMP70, GFP-PMP70(AA.263-375), including TMD5 and TMD6, exhibited the peroxisomal localization. PMP70(AA.263-375) also possesses hydrophobic residues (Ile307/Leu308) in the region adjacent to TMD5, which were important for targeting. These results suggest that PMP70 possesses two distinct targeting signals, and hydrophobic regions adjacent to the first TMD of each region are important for targeting.

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