Lumenal Endosomal and Golgi-Retrieval Determinants Involved in pH-sensitive Targeting of an Early Golgi Protein

Despite the potential importance of retrieval-based targeting, few Golgi cisternae-localized proteins have been demonstrated to be targeted by retrieval, and the putative retrieval signals remain unknown. Golgi phosphoprotein of 130 kDa (GPP130) is acis-Golgi protein that allows assay of retrieval-based targeting because it redistributes to endosomes upon treatment with agents that disrupt lumenal pH, and it undergoes endosome-to-Golgi retrieval upon drug removal. Analysis of chimeric molecules containing domains from GPP130 and the plasma membrane protein dipeptidylpeptidase IV indicated that GPP130 targeting information is contained entirely within its lumenal domain. Dissection of the lumenal domain indicated that a predicted coiled-coil stem domain adjacent to the transmembrane domain was both required and sufficient for pH-sensitive Golgi localization and endosome-to-Golgi retrieval. Further dissection of this stem domain revealed two noncontiguous stretches that each conferred Golgi localization separated by a stretch that conferred endosomal targeting. Importantly, in the absence of the endosomal determinant the Golgi targeting of constructs containing either or both of the Golgi determinants became insensitive to pH disruption by monensin. Because monensin blocks endosome-to-Golgi transport, the finding that the endosomal determinant confers monensin sensitivity suggests that the endosomal determinant causes GPP130 to traffic to endosomes from which it is normally retrieved. Thus, our observations identify Golgi and endosomal targeting determinants within a lumenal predicted coiled-coil domain that appear to act coordinately to mediate retrieval-based targeting of GPP130.

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Cryo-EM structures of human TMEM120A and TMEM120B

10.1101/2021.06.27.450060 ◽

2021 ◽

Author(s):

Meng Ke ◽

Yue Yu ◽

Changjian Zhao ◽

Shirong Lai ◽

Qiang Su ◽

...

Keyword(s):

Potential Role ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Expression System ◽

Coiled Coil ◽

Fatty Acid Elongase ◽

Coiled Coil Domain ◽

Induced Currents ◽

The Difference

TMEM120A (Transmembrane protein 120A) was recently identified as a mechanical pain sensing ion channel named as TACAN, while its homologue TMEM120B has no mechanosensing property1. Here, we report the cryo-EM structures of both human TMEM120A and TMEM120B. The two structures share the same dimeric assembly, mediated by extensive interactions through the transmembrane domain (TMD) and the N-terminal coiled coil domain (CCD). However, the nearly identical structures cannot provide clues for the difference in mechanosensing between TMEM120A and TMEM120B. Although TMEM120A could mediate conducting currents in a bilayer system, it does not mediate mechanical-induced currents in a heterologous expression system, suggesting TMEM120A is unlikely a mechanosensing channel. Instead, the TMDs of TMEM120A and TMEM120B resemble the structure of a fatty acid elongase, ELOVL7, indicating their potential role of an enzyme in lipid metabolism.

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The Membrane Transport Factor TAP/p115 Cycles between the Golgi and Earlier Secretory Compartments and Contains Distinct Domains Required for Its Localization and Function

The Journal of Cell Biology ◽

10.1083/jcb.143.2.319 ◽

1998 ◽

Vol 143 (2) ◽

pp. 319-331 ◽

Cited By ~ 102

Author(s):

David S. Nelson ◽

Cecilia Alvarez ◽

Ya-sheng Gao ◽

Rafael García-Mata ◽

Elizabeth Fialkowski ◽

...

Keyword(s):

Binding Domain ◽

Terminal Region ◽

Transfected Cells ◽

Acidic Domain ◽

Golgi Transport ◽

Golgi Localization ◽

Interphase Cells ◽

Golgi Protein ◽

And Function ◽

Mammalian Protein

The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al., 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15°C treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi. TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.

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The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP.

The Journal of Cell Biology ◽

10.1083/jcb.131.4.895 ◽

1995 ◽

Vol 131 (4) ◽

pp. 895-912 ◽

Cited By ~ 124

Author(s):

S Schröder ◽

F Schimmöller ◽

B Singer-Krüger ◽

H Riezman

Keyword(s):

Saccharomyces Cerevisiae ◽

Steady State ◽

Transmembrane Protein ◽

Retrograde Transport ◽

Type I ◽

Hybrid Protein ◽

Plasma Membrane Protein ◽

Golgi Localization ◽

Golgi Compartment ◽

Golgi Protein

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge-independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p-Emp47p hybrid protein was mislocalized to the cell surface in the alpha-COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.

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Sorting of yeast alpha 1,3 mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted protein.

Molecular Biology of the Cell ◽

10.1091/mbc.6.7.809 ◽

1995 ◽

Vol 6 (7) ◽

pp. 809-824 ◽

Cited By ~ 46

Author(s):

T R Graham ◽

V A Krasnov

Keyword(s):

Golgi Complex ◽

Fusion Proteins ◽

Transmembrane Domain ◽

Signal Sequence ◽

Integral Membrane Protein ◽

Retention Mechanism ◽

Secreted Protein ◽

Golgi Localization ◽

In The Wild ◽

Lumenal Domain

alpha 1,3 mannosyltransferase (Mnn1p) is a type II integral membrane protein that is localized to the yeast Golgi complex. We have examined the signals within Mnn1p that mediate Golgi localization by expression of fusion proteins comprised of Mnn1p and the secreted protein invertase. The N-terminal transmembrane domain (TMD) of Mnn1p is sufficient to localize invertase to the Golgi complex by a mechanism that is not saturable by approximately 15-20 fold overexpression. Furthermore, the TMD-mediated localization mechanism is clathrin dependent, as an invertase fusion protein bearing only the Mnn1p TMD is mislocalized to the plasma membrane of a clathrin heavy chain mutant. The Mnn1-invertase fusion proteins are not retained in the Golgi complex as efficiently as Mnn1p, suggesting that other signals may be present in the wild-type protein. Indeed, the Mnn1p lumenal domain (Mnn1-s) is also localized to the Golgi complex when expressed as a functional, soluble protein by exchanging its TMD for a cleavable signal sequence. In contrast to the Mnn1-invertase fusion proteins, overexpression of Mnn1-s saturates its retention mechanism, and results in the partial secretion of this protein. These data indicate that Mnn1p has separable Golgi localization signals within both its transmembrane and lumenal domains.

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The coiled-coil domain of glycosomal membrane-associated Leishmania donovani PEX14: cloning, overexpression, purification and preliminary crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20011127 ◽

2020 ◽

Vol 76 (10) ◽

pp. 464-468

Author(s):

Anil Kumar Shakya ◽

J. Venkatesh Pratap

Keyword(s):

Leishmania Donovani ◽

Protein Import ◽

Transmembrane Domain ◽

Coiled Coil ◽

Supramolecular Assembly ◽

Crystallographic Analysis ◽

Size Exclusion ◽

Monoclinic Space Group ◽

Cell Parameters ◽

Coiled Coil Domain

The glycosomal membrane-associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chromatography. Crystals that diffracted to 1.98 Å resolution were obtained from a condition consisting of 1.4 M sodium citrate tribasic dihydrate, 0.1 M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 143.98, b = 32.62, c = 95.62 Å, β = 94.68°. Structure determination and characterization are in progress.

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A Role for the Lumenal Domain in Golgi Localization of theSaccharomyces cerevisiaeGuanosine Diphosphatase

Molecular Biology of the Cell ◽

10.1091/mbc.9.6.1351 ◽

1998 ◽

Vol 9 (6) ◽

pp. 1351-1365 ◽

Cited By ~ 16

Author(s):

Jennifer J. Vowels ◽

Gregory S. Payne

Keyword(s):

Protein Interactions ◽

Transmembrane Domain ◽

Chimeric Protein ◽

Cytoplasmic Domain ◽

Type Ii ◽

Protein Protein Interactions ◽

Amino Terminal ◽

Golgi Localization ◽

Vacuolar Protein ◽

Lumenal Domain

Integral membrane proteins (IMPs) contain localization signals necessary for targeting to their resident subcellular compartments. To define signals that mediate localization to the Golgi complex, we have analyzed a resident IMP of the Saccharomyces cerevisiaeGolgi complex, guanosine diphosphatase (GDPase). GDPase, which is necessary for Golgi-specific glycosylation reactions, is a type II IMP with a short amino-terminal cytoplasmic domain, a single transmembrane domain (TMD), and a large catalytic lumenal domain. Regions specifying Golgi localization were identified by analyzing recombinant proteins either lacking GDPase domains or containing corresponding domains from type II vacuolar IMPs. Neither deletion nor substitution of the GDPase cytoplasmic domain perturbed Golgi localization. Exchanging the GDPase TMD with vacuolar protein TMDs only marginally affected Golgi localization. Replacement of the lumenal domain resulted in mislocalization of the chimeric protein from the Golgi to the vacuole, but a similar substitution leaving 34 amino acids of the GDPase lumenal domain intact was properly localized. These results identify a major Golgi localization determinant in the membrane-adjacent lumenal region (stem) of GDPase. Although necessary, the stem domain is not sufficient to mediate localization; in addition, a membrane-anchoring domain and either the cytoplasmic or full-length lumenal domain must be present to maintain Golgi residence. The importance of lumenal domain sequences in GDPase Golgi localization and the requirement for multiple hydrophilic protein domains support a model for Golgi localization invoking protein–protein interactions rather than interactions between the TMD and the lipid bilayer.

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Generation of a fusion protein containing the two functional coiled-coil domain of t- SNARE, SNAP-23 and a transmembrane domain for mast cell

Journal of Applied and Natural Science ◽

10.31018/jans.v12i4.2439 ◽

2020 ◽

Vol 12 (4) ◽

pp. 670-674

Author(s):

D. M. Agase ◽

S. B. Zade ◽

T.S. Kothe

Keyword(s):

Mast Cell ◽

Fusion Protein ◽

Membrane Fusion ◽

Transmembrane Domain ◽

Coiled Coil ◽

System A ◽

Coiled Coil Domain ◽

Novel Approach ◽

Protein Receptor ◽

Cellular Machinery

SNAREs (Soluble N-Ethylmaleimide-Sensitive Fusion Protein Attachment Protein Receptor) are a class of membrane proteins that mediate membrane-membrane fusion in eukaryotic cells. SNAP-23 is a t-SNARE which is a component of cellular machinery is required for membrane fusion. SNAP-23 lacks transmembrane domain. Cysteines in the linker region of SNAP-23 are involved in targeting of SNAP-23 to the membrane. In the present work, a portion of MDR3 gene (MDR3 1-145) and CLP24 (CLP134-195) was subcloned into a plasmid encoding EGFP-SNAP-23 Cys- mutant for the generation of a fusion protein containing the two functional coiled-coil domain of t-SNARE, SNAP 23 and a transmembrane domain of MDR3 gene and CLP24 for mast cell. This fusion protein will be important to study the membrane targeting and raft association of the chimeric SNAP23 protein, which plays an important role in mast cell exocytosis in the mammalian system. A novel bioinformatics approach has been applied to identify the specific transmembrane domain. This novel approach can be used to construct other fusion proteins.

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TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

Biochemical Journal ◽

10.1042/bcj20190359 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3241-3260

Author(s):

Sindhu Wisesa ◽

Yasunori Yamamoto ◽

Toshiaki Sakisaka

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Membrane Protein ◽

Mammalian Cells ◽

Coiled Coil ◽

Transmembrane Domains ◽

Domain Family ◽

Coiled Coil Domain ◽

U2os Cells ◽

Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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