scholarly journals Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi to Plasma Membrane Transport Intermediates in Living Cells

1998 ◽  
Vol 143 (6) ◽  
pp. 1485-1503 ◽  
Author(s):  
Koret Hirschberg ◽  
Chad M. Miller ◽  
Jan Ellenberg ◽  
John F. Presley ◽  
Eric D. Siggia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Transport ◽  
Golgi Complex ◽  
Rate Constants ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Single Cells ◽  
Cell Periphery ◽  
Protein Traffic ◽  
Golgi Transport

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments. The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG– GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG–GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG–GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.

scholarly journals Oligomerization of a membrane protein correlates with its retention in the Golgi complex

1993 ◽  
Vol 122 (6) ◽  
pp. 1185-1196 ◽  
Author(s):  
OA Weisz ◽  
AM Swift ◽  
CE Machamer
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Cytochalasin D ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Proteolytic Digestion ◽  
Golgi Transport ◽  
Large Oligomer ◽  
Membrane Spanning ◽  
Lumenal Domain

The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.

Transport Through the Secretory Pathway of VSVG Tagged With Green Fluorescent Protein: Role of Tubulovesicular Carriers and Microtubules

1997 ◽  
Vol 3 (S2) ◽  
pp. 139-140
Author(s):  
John Presley ◽  
Koret Hirschberg ◽  
Nelson Cole
Keyword(s):  
Green Fluorescent Protein ◽  
Membrane Transport ◽  
G Protein ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dynamic Properties ◽  
Living Cells ◽  
Morphological Properties ◽  
Green Fluorescent

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.

scholarly journals Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps.

1995 ◽  
Vol 131 (6) ◽  
pp. 1421-1433 ◽  
Author(s):  
P A Conrad ◽  
E J Smart ◽  
Y S Ying ◽  
R G Anderson ◽  
G S Bloom
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Cholesterol Oxidase ◽  
Human Fibroblasts ◽  
Subsequent Removal ◽  
Golgi Transport ◽  
Cytoplasmic Face ◽  
Block Movement ◽  
Intermediate Compartment ◽  
In Cells

Caveolin is a protein associated with the characteristic coats that decorate the cytoplasmic face of plasma membrane caveolae. Recently it was found that exposure of human fibroblasts to cholesterol oxidase (CO) rapidly induces caveolin to redistribute to the ER and then to the Golgi complex, and that subsequent removal of CO allows caveolin to return to the plasma membrane (Smart, E. J., Y.-S. Ying, P. A. Conrad, R. G. W. Anderson, J. Cell Biol. 1994, 127:1185-1197). We now present evidence that caveolin normally undergoes microtubule-dependent cycling between the plasma membrane and the Golgi. In cells that were treated briefly with nocodazole and then with a mixture of nocodazole plus CO, caveolin relocated from the plasma membrane to the ER and then to the ER/Golgi intermediate compartment (ERGIC), but subsequent movement to the Golgi was not observed. Even in the absence of CO, nocodazole caused caveolin to accumulate in the ERGIC. Nocodazole did not retard the movement of caveolin from the Golgi to the plasma membrane after removal of CO. Incubation of cells at 15 degrees followed by elevation of the temperature to 37 degrees caused caveolin to accumulate first in the ERGIC and then in the Golgi, before finally reestablishing its normal steady state distribution predominantly in plasma membrane caveolae. In cells released from a 15 degrees block, movement of caveolin from the Golgi to the plasma membrane was not inhibited by nocodazole. Taken together, these results imply that caveolin cycles constitutively between the plasma membrane and the Golgi by a multi-step process, one of which, ERGIC-to-Golgi transport, requires microtubules. This novel, bidirectional pathway may indicate roles for microtubules in the maintenance of caveolae, and for caveolin in shuttling fatty acids and cholesterol between the plasma membrane and the ER/Golgi system.

Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation

2011 ◽  
Vol 437 (3) ◽  
pp. 389-397 ◽  
Author(s):  
Bastian Thaa ◽  
Ilya Levental ◽  
Andreas Herrmann ◽  
Michael Veit
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Glutathione Transferase ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Yellow Fluorescent Protein ◽  
Cytoplasmic Tail ◽  
Proton Channel ◽  
Amphiphilic Helix ◽  
Cholesterol Binding

The influenza virus transmembrane protein M2 is a proton channel, but also plays a role in the scission of nascent virus particles from the plasma membrane. An amphiphilic helix in the CT (cytoplasmic tail) of M2 is supposed to insert into the lipid bilayer, thereby inducing curvature. Palmitoylation of the helix and binding to cholesterol via putative CRAC (cholesterol recognition/interaction amino acid consensus) motifs are believed to target M2 to the edge of rafts, the viral-budding site. In the present study, we tested pre-conditions of this model, i.e. that the CT interacts with membranes, and that acylation and cholesterol binding affect targeting of M2. M2-CT, purified as a glutathione transferase fusion protein, associated with [3H]photocholesterol and with liposomes. Mutation of tyrosine residues in the CRAC motifs prevented [3H]photocholesterol labelling and reduced liposome binding. M2-CT fused to the yellow fluorescent protein localized to the Golgi in transfected cells; membrane targeting was dependent on CRAC and (to a lesser extent) on palmitoylation. Preparation of giant plasma membrane vesicles from cells expressing full-length M2–GFP (green fluorescent protein) showed that the protein is partly present in the raft domain. Raft targeting required palmitoylation, but not the CRAC motifs. Thus palmitoylation and cholesterol binding differentially affect the intrinsic membrane binding of the amphiphilic helix.

scholarly journals Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction

2019 ◽  
Vol 30 (24) ◽  
pp. 2996-3012 ◽  
Author(s):  
Soyeon Kim ◽  
Joseph M. Kalappurakkal ◽  
Satyajit Mayor ◽  
Michael K. Rosen
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Transmembrane Protein ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Basal Plasma Membrane ◽  
Basal Plasma ◽  
Multivalent Interactions ◽  
And Control ◽  
Actomyosin Contraction

The plasma membrane of eukaryotic cells is organized into lipid and protein microdomains, whose assembly mechanisms and functions are incompletely understood. We demonstrate that proteins in the nephrin/Nck/N-WASP actin-regulatory pathway cluster into micron-scale domains at the basal plasma membrane upon triggered phosphorylation of transmembrane protein nephrin. The domains are persistent but readily exchange components with their surroundings, and their formation is dependent on the number of Nck SH3 domains, suggesting they are phase separated polymers assembled through multivalent interactions among the three proteins. The domains form independent of the actin cytoskeleton, but acto-myosin contractility induces their rapid lateral movement. Nephrin phosphorylation induces larger clusters at the cell periphery, which are associated with extensive actin assembly and dense filopodia. Our studies illustrate how multivalent interactions between proteins at the plasma membrane can produce micron-scale organization of signaling molecules, and how the resulting clusters can both respond to and control the actin cytoskeleton.

scholarly journals A Novel Type III Endosome Transmembrane Protein, TEMP

Cells ◽  
2012 ◽  
Vol 1 (4) ◽  
pp. 1029-1044 ◽  
Author(s):  
Rajith N. Aturaliya ◽  
Markus C. Kerr ◽  
Rohan D. Teasdale
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Subcellular Localisation ◽  
Cell Periphery ◽  
Recycling Endosomes ◽  
Type Iii ◽  
Extracellular Region ◽  
Early Endosomes

As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP's plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport.

scholarly journals Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic Events

2001 ◽  
Vol 12 (3) ◽  
pp. 601-614 ◽  
Author(s):  
Dennis C. Ko ◽  
Michael D. Gordon ◽  
Janet Y. Jin ◽  
Matthew P. Scott
Keyword(s):  
High Speed ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Density Lipoprotein ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Late Endosomes ◽  
Npc1 Gene ◽  
Niemann Pick ◽  
Mutant Cells

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. InNPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.

scholarly journals Simultaneous visualization of the translocation of protein kinase Cα–green fluorescent protein hybrids and intracellular calcium concentrations

1999 ◽  
Vol 337 (2) ◽  
pp. 211-218 ◽  
Author(s):  
Kasper ALMHOLT ◽  
Per O. G. ARKHAMMAR ◽  
Ole THASTRUP ◽  
S⊘ren TULLIN
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Protein Kinase ◽  
Intracellular Calcium ◽  
Time Course ◽  
Fluorescent Protein ◽  
Single Cells ◽  
High Concentrations ◽  
Green Fluorescent ◽  
Simultaneous Visualization

The α isoform of protein kinase C (PKCα) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKCα was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKCα in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+]i) and PKCα translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+]i was observed, without detectable translocation of PKCα. In contrast, PMA induced translocation of PKCα without any increase in [Ca2+]i. Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKCα, indicating that PKCα translocation may be independent of both actin filaments and microtubules. The time course of PKCα translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.

scholarly journals A Tyrosine-Based Trafficking Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary Envelopment

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Andrea N. Dietz ◽  
Clarissa Villinger ◽  
Stefan Becker ◽  
Manfred Frick ◽  
Jens von Einem
Keyword(s):  
Plasma Membrane ◽  
Antiviral Therapy ◽  
Human Cytomegalovirus ◽  
Golgi Complex ◽  
Fluorescent Protein ◽  
Treatment Options ◽  
Intracellular Localization ◽  
Tegument Protein ◽  
Genome Replication ◽  
Virus Infections

ABSTRACTThe human cytomegalovirus (HCMV) tegument protein pUL71 is required for efficient secondary envelopment and accumulates at the Golgi compartment-derived viral assembly complex (vAC) during infection. Analysis of various C-terminally truncated pUL71 proteins fused to enhanced green fluorescent protein (eGFP) identified amino acids 23 to 34 as important determinants for its Golgi complex localization. Sequence analysis and mutational verification revealed the presence of an N-terminal tyrosine-based trafficking motif (YXXΦ) in pUL71. This led us to hypothesize a requirement of the YXXΦ motif for the function of pUL71 in infection. Mutation of both the tyrosine residue and the entire YXXΦ motif resulted in an altered distribution of mutant pUL71 at the plasma membrane and in the cytoplasm during infection. Both YXXΦ mutant viruses exhibited similarly decreased focal growth and reduced virus yields in supernatants. Ultrastructurally, mutant-virus-infected cells exhibited impaired secondary envelopment manifested by accumulations of capsids undergoing an envelopment process. Additionally, clusters of capsid accumulations surrounding the vAC were observed, similar to the ultrastructural phenotype of a UL71-deficient mutant. The importance of endocytosis and thus the YXXΦ motif for targeting pUL71 to the Golgi complex was further demonstrated when clathrin-mediated endocytosis was inhibited either by coexpression of the C-terminal part of cellular AP180 (AP180-C) or by treatment with methyl-β-cyclodextrin. Both conditions resulted in a plasma membrane accumulation of pUL71. Altogether, these data reveal the presence of a functional N-terminal endocytosis motif that is an important determinant for intracellular localization of pUL71 and that is furthermore required for the function of pUL71 during secondary envelopment of HCMV capsids at the vAC.IMPORTANCEHuman cytomegalovirus (HCMV) is the leading cause of birth defects among congenital virus infections and can lead to life-threatening infections in immunocompromised hosts. Current antiviral treatments target viral genome replication and are increasingly overcome by viral mutations. Therefore, identifying new targets for antiviral therapy is important for future development of novel treatment options. A detailed molecular understanding of the complex virus morphogenesis will identify potential viral as well as cellular targets for antiviral intervention. Secondary envelopment is an important viral process through which infectious virus particles are generated and which involves the action of several viral proteins, such as tegument protein pUL71. Targeting of pUL71 to the site of secondary envelopment appears to be crucial for its function during this process and is regulated by utilizing host trafficking mechanisms that are commonly exploited by viral glycoproteins. Thus, intracellular trafficking, if targeted, might present a novel target for antiviral therapy.

scholarly journals Palmitoylation of the oncogenic RhoGEF TGAT is dispensable for membrane localization and consequent activation of RhoA

2016 ◽  
Author(s):  
Jakobus van Unen ◽  
Dennis Botman ◽  
Taofei Yin ◽  
Yi I. Wu ◽  
Mark A. Hink ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Subcellular Location ◽  
Membrane Localization ◽  
Membrane Association ◽  
Fluorescence Signal ◽  
Wild Type ◽  
Cellular Cytoskeleton ◽  
And Control

AbstractRho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and thereby regulate and control processes such as cell migration, cell adhesion and proliferation. TGAT is a splice variant of the RhoGEF Trio, with oncogenic potential. Whether the subcellular location of TGAT is critical for its activity is unknown. Confocal microscopy of fluorescent protein tagged TGAT revealed co-localization with a Golgi marker. Because plasma membrane localized RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was studied. In order to quantitatively measure plasma membrane association we developed a novel, highly sensitive image analysis method. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGATΔ15, lost membrane association. The functional role of membrane localization was determined by measuring TGAT activity in single cells with a RhoA FRET-sensor and F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGATΔ15 variant. Synthetic recruitment of TGATΔ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Together, these results show that membrane association of TGAT is critical for its activity, but that palmitoylation is dispensable.

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