A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells

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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Clathrin- and Arp2/3-Independent Endocytosis in the Fungal Pathogen Candida albicans

mBio ◽

10.1128/mbio.00476-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 20

Author(s):

Elias Epp ◽

Elena Nazarova ◽

Hannah Regan ◽

Lois M. Douglas ◽

James B. Konopka ◽

...

Keyword(s):

Candida Albicans ◽

Membrane Proteins ◽

Fungal Pathogen ◽

Mammalian Cells ◽

Fluid Phase ◽

Fungal Species ◽

Endocytic Pathway ◽

Dependent Manner ◽

Content Type ◽

Actin Cables

ABSTRACT Clathrin-mediated endocytosis (CME) is conserved among eukaryotes and has been extensively analyzed at a molecular level. Here, we present an analysis of CME in the human fungal pathogen Candida albicans that shows the same modular structure as those in other fungi and mammalian cells. Intriguingly, C. albicans is perfectly viable in the absence of Arp2/3, an essential component of CME in other systems. In C. albicans, Arp2/3 function remains essential for CME as all 15 proteins tested that participate in CME, including clathrin, lose their characteristic dynamics observed in wild-type (WT) cells. However, since arp2/3 cells are still able to endocytose lipids and fluid-phase markers, but not the Ste2 and Mup1 plasma membrane proteins, there must be an alternate clathrin-independent pathway we term Arp2/3-independent endocytosis (AIE). Characterization of AIE shows that endocytosis in arp2 mutants relies on actin cables and other Arp2/3-independent actin structures, as inhibition of actin functions prevented cargo uptake in arp2/3 mutants. Transmission electron microscopy (TEM) showed that arp2/3 mutants still formed invaginating tubules, cell structures whose proper functions are believed to heavily rely on Arp2/3. Finally, Prk1 and Sjl2, two proteins involved in patch disassembly during CME, were not correctly localized to sites of endocytosis in arp2 mutants, implying a role of Arp2/3 in CME patch disassembly. Overall, C. albicans contains an alternative endocytic pathway (AIE) that relies on actin cable function to permit clathrin-independent endocytosis (CIE) and provides a system to further explore alternate endocytic routes that likely exist in fungal species. IMPORTANCE There is a well-established process of endocytosis that is generally used by eukaryotic cells termed clathrin-mediated endocytosis (CME). Although the details are somewhat different between lower and higher eukaryotes, CME appears to be the dominant endocytic process in all eukaryotes. While fungi such as Saccharomyces cerevisiae have proven excellent models for dissecting the molecular details of endocytosis, loss of CME is so detrimental that it has been difficult to study alternate pathways functioning in its absence. Although the fungal pathogen Candida albicans has a CME pathway that functions similarly to that of S. cerevisiae, inactivation of this pathway does not compromise growth of yeast-form C. albicans. In these cells, lipids and fluid-phase molecules are still endocytosed in an actin-dependent manner, but membrane proteins are not. Thus, C. albicans provides a powerful model for the analysis of CME-independent endocytosis in lower eukaryotes.

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Evidence that the entire Golgi apparatus cycles in interphase HeLa cells

The Journal of Cell Biology ◽

10.1083/jcb.200103104 ◽

2001 ◽

Vol 155 (4) ◽

pp. 543-556 ◽

Cited By ~ 73

Author(s):

Suzanne Miles ◽

Heather McManus ◽

Kimberly E. Forsten ◽

Brian Storrie

Keyword(s):

Membrane Proteins ◽

Golgi Apparatus ◽

Mammalian Cells ◽

Dynamic Structure ◽

Dominant Negative ◽

Integral Membrane Proteins ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Golgi Region ◽

Exit Block

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus–like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1pdn concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region–integral membrane proteins cycle through ER in interphase cells.

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Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/s1388-1981(99)00137-7 ◽

1999 ◽

Vol 1441 (1) ◽

pp. 4-13 ◽

Cited By ~ 168

Author(s):

N Abumrad

Keyword(s):

Fatty Acid ◽

Membrane Proteins ◽

Mammalian Cells ◽

Fatty Acid Uptake ◽

Chain Fatty Acid ◽

Acid Uptake ◽

Long Chain Fatty Acid ◽

Long Chain

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Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

The Journal of Cell Biology ◽

10.1083/jcb.137.6.1199 ◽

1997 ◽

Vol 137 (6) ◽

pp. 1199-1210 ◽

Cited By ~ 160

Author(s):

Li Yang ◽

Tinglu Guan ◽

Larry Gerace

Keyword(s):

Membrane Proteins ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Mammalian Cells ◽

Polyclonal Antibodies ◽

Integral Membrane Proteins ◽

Immunogold Electron Microscopy ◽

Nuclear Pore ◽

Nuclear Lamins ◽

Nuclear Membranes

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC6 and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.

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Role of Ubiquitylation in Cellular Membrane Transport

Physiological Reviews ◽

10.1152/physrev.00020.2005 ◽

2006 ◽

Vol 86 (2) ◽

pp. 669-707 ◽

Cited By ~ 162

Author(s):

Olivier Staub ◽

Daniela Rotin

Keyword(s):

Membrane Proteins ◽

Mammalian Cells ◽

Secretory Pathway ◽

Surface Expression ◽

Cellular Membrane ◽

Cell Surface Expression ◽

Multivesicular Bodies ◽

The Past ◽

Endoplasmic Reticulum Associated Degradation

Ubiquitylation of membrane proteins has gained considerable interest in recent years. It has been recognized as a signal that negatively regulates the cell surface expression of many plasma membrane proteins both in yeast and in mammalian cells. Moreover, it is also involved in endoplasmic reticulum-associated degradation of membrane proteins, and it acts as a sorting signal both in the secretory pathway and in endosomes, where it targets proteins into multivesicular bodies in the lumen of vacuoles/lysosomes. In this review we discuss the progress in understanding these processes, achieved during the past several years.

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Characterization of constitutive ER-phagy of excess membrane proteins

PLoS Genetics ◽

10.1371/journal.pgen.1009255 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009255

Author(s):

Zhanna Lipatova ◽

Valeriya Gyurkovska ◽

Sarah F. Zhao ◽

Nava Segev

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Human Health ◽

Mammalian Cells ◽

Normal Growth ◽

Integral Membrane Proteins ◽

Yeast Cells ◽

Cellular Proteins

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

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Molecular Basis of Oligoubiquitin-Dependent Internalization of Membrane Proteins in Mammalian Cells

Traffic ◽

10.1111/j.1600-0854.2006.00384.x ◽

2006 ◽

Vol 7 (3) ◽

pp. 282-297 ◽

Cited By ~ 91

Author(s):

Herve Barriere ◽

Csilla Nemes ◽

Delphine Lechardeur ◽

Mina Khan-Mohammad ◽

Klaus Fruh ◽

...

Keyword(s):

Membrane Proteins ◽

Molecular Basis ◽

Mammalian Cells

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Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2009.0179 ◽

2010 ◽

Vol 365 (1541) ◽

pp. 775-784 ◽

Cited By ~ 72

Author(s):

Roberto Docampo ◽

Paul Ulrich ◽

Silvia N. J. Moreno

Keyword(s):

Membrane Proteins ◽

Mammalian Cells ◽

Physiological Function ◽

Morphological Characteristics ◽

Contractile Vacuole ◽

Ph Homeostasis ◽

Acidic Properties ◽

Eukaryotic Microbes ◽

Lysosome Related Organelles

Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca 2+ -ATPase, V-H + -ATPase, H + -PPase), exchangers (Na + /H + , Ca 2+ /H + ), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes.

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