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ABSTRACT: Lipid droplets form the storage reservoirs for lipids in adipocytes, and their stable appearance suggests a static nature of lipid storage. A stable lipid store, however, may be maintained through the dynamic recycling of lipid cargo between the cytoplasmic compartment and the lipid droplet. In this study, we applied live-cell microscopy to follow intracellular transport steps of fluorescently labeled fatty acids in differentiated 3T3-L1 adipocytes. We demonstrate that intracellular lipids continuously exit and re-enter lipid droplets, and that individual lipid droplets exchange their content on a timescale of minutes. These data demonstrate a surprisingly high rate of intracellular lipid turnover in adipocytes and support the novel concept that lipid storage is achieved by dynamic recycling rather than static retention.
FEBS letters 06/2011; 585(12):1946-50. · 3.54 Impact Factor
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ABSTRACT: Increased body fat correlates with the enlargement of average fat cell size and reduced adipose tissue insulin sensitivity. It is currently unclear whether adipocytes, as they accumulate more triglycerides and grow in size, gradually become less insulin sensitive or whether obesity-related factors independently cause both the enlargement of adipocyte size and reduced adipose tissue insulin sensitivity. In the first instance, large and small adipocytes in the same tissue would exhibit differences in insulin sensitivity, whereas, in the second instance, adipocyte size per se would not necessarily correlate with insulin response. To analyze the effect of adipocyte size on insulin sensitivity, we employed a new single-cell imaging assay that resolves fatty acid uptake and insulin response in single adipocytes in subcutaneous adipose tissue explants. Here, we report that subcutaneous adipocytes are heterogeneous in size and intrinsic insulin sensitivity. Whereas smaller adipocytes respond to insulin by increasing lipid uptake, adipocytes with cell diameters larger than 80-100 microm are insulin resistant. We propose that, when cell size approaches a critical boundary, adipocytes lose insulin-dependent fatty acid transport. This negative feedback mechanism may protect adipocytes from lipid overload and restrict further expansion of adipose tissue, which leads to obesity and metabolic complications.
AJP Endocrinology and Metabolism 09/2010; 299(3):E486-96. · 4.75 Impact Factor
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ABSTRACT: Localization of a membrane protein in a subcellular compartment can be achieved by its retention in the compartment or by its continuous transport toward this compartment. Previous results have suggested that specific enzymes are localized in the Golgi apparatus at least in part by selective retention and exclusion from transport vesicles. However, the function of some Golgi SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins is not compatible with their exclusion from transport vesicles. To help understand the mechanism accounting for the localization of SNARE proteins in the Golgi apparatus, we analyzed their lateral distribution in the Golgi cisternae and their incorporation into transport vesicles. According to our results, all SNARE proteins are efficiently incorporated into transport vesicles, indicating that the localization of SNARE proteins in the Golgi apparatus is not based on a static retention mechanism. Detailed analysis suggested that incorporation into transport vesicles was more efficient for SNARE proteins restricted to the cis face of the Golgi as compared with SNAREs present at the trans face. Furthermore, overexpression of a cis-Golgi SNARE protein altered concomitantly its incorporation in transport vesicles and its intra-Golgi localization. These observations suggest that, contrary to resident Golgi enzymes, SNARE proteins are localized in the Golgi apparatus as the result of a dynamic transport equilibrium.
Proceedings of the National Academy of Sciences 11/2005; 102(41):14647-52. · 9.68 Impact Factor
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Allen Volchuk,
Mariella Ravazzola,
Alain Perrelet,
William S Eng,
Maurizio Di Liberto, Oleg Varlamov,
Masayoshi Fukasawa,
Thomas Engel,
Thomas H Söllner,
James E Rothman,
Lelio Orci
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ABSTRACT: Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion.
Molecular Biology of the Cell 05/2004; 15(4):1506-18. · 4.94 Impact Factor
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ABSTRACT: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze compartment-specific membrane fusion. Whereas most SNAREs are bona fide type II membrane proteins, Ykt6 lacks a proteinaceous membrane anchor but contains a prenylation consensus motif (CAAX box) and exists in an inactive cytosolic and an active membrane-bound form. We demonstrate that both forms are farnesylated at the carboxyl-terminal cysteine of the CCAIM sequence. Farnesylation is the prerequisite for subsequent palmitoylation of the upstream cysteine, which permits stable membrane association of Ykt6. The double-lipid modification and membrane association is crucial for intra-Golgi transport in vitro and cell homeostasis/survival in vivo. The membrane recruitment and palmitoylation is controlled by the N-terminal domain of Ykt6, which interacts with the SNARE motif, keeping it in an inactive closed conformation. Together, these results suggest that conformational changes control the lipid modification and function of Ykt6. Considering the essential and central role of Ykt6 in the secretory pathway, this spatial and functional cycle might provide a mechanism to regulate the rate of intracellular membrane flow.
Proceedings of the National Academy of Sciences 05/2004; 101(14):4815-20. · 9.68 Impact Factor
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Oleg Varlamov,
Allen Volchuk,
Vahid Rahimian,
Claudia A Doege,
Fabienne Paumet,
William S Eng,
Nancy Arango,
Francesco Parlati,
Mariella Ravazzola,
Lelio Orci,
Thomas H Söllner,
James E Rothman
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ABSTRACT: A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.
The Journal of Cell Biology 02/2004; 164(1):79-88. · 10.26 Impact Factor
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Oleg Varlamov,
Allen Volchuk,
Vahid Rahimian,
Claudia A. Doege,
Fabienne Paumet,
William S. Eng,
Nancy Arango,
Francesco Parlati,
Mariella Ravazzola,
Lelio Orci,
Thomas H. Söllner,
James E. Rothman
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ABSTRACT: A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by
substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were
tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions.
A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated
by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves
could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by
the complementary countercurrent distributions of the i-SNAREs.
The Journal of Cell Biology 01/2004; 164(1):79-88. · 10.26 Impact Factor
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ABSTRACT: SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells. Sequence analysis of the yeast genome revealed a previously uncharacterized SNARE, SNARE-like tail-anchored protein 1 (Slt1). Slt1 is an essential protein localized in the endoplasmic reticulum (ER). It forms a SNARE complex with Sec22 and the ER syntaxin Ufe1. Down-regulation of Slt1 levels leads to improper secretion of proteins normally resident in the ER. We suggest that Slt1 is a component of the SNAREpin required for retrograde traffic to the ER. Based on the previously reported association with Ufe1 and Sec22, Sec20 likely contributes the fourth SNARE to the SNAREpin.
Proceedings of the National Academy of Sciences 09/2003; 100(17):9873-7. · 9.68 Impact Factor
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ABSTRACT: Syntaxin-5 (Sed5) is the only syntaxin needed for transport into and across the yeast Golgi, raising the question of how a single syntaxin species could mediate vesicle transport in both the anterograde and the retrograde direction within the stack. Sed5 is known to combine with two light chains (Bos1 and Sec22) to form the t-SNARE needed to receive vesicles from the endoplasmic reticulum. However, the yeast Golgi contains several other potential light chains with which Sed5 could potentially combine to form other t-SNAREs. To explore the degree of specificity in the choice of light chains by a t-SNARE, we undertook a comprehensive examination of the capacity of all 21 Sed5-based t-SNAREs that theoretically could assemble in the yeast Golgi to fuse with each of the 7 potential v-SNAREs also present in this organelle. Only one additional of these 147 combinations was fusogenic. This functional proteomic strategy thereby revealed a previously uncharacterized t-SNARE in which Sed5 is the heavy chain and Gos1 and Ykt6 are the light chains, and whose unique cognate v-SNARE is Sft1. Immunoprecipitation experiments confirmed the existence of this complex in vivo. Fusion mediated by this second Golgi SNAREpin is topologically restricted, and existing genetic and morphologic evidence implies that it is used for transport across the Golgi stack. From this study, together with the previous functional proteomic analyses which have tested 275 distinct quaternary SNARE combinations, it follows that the fusion potential and transport pathways of the yeast cell can be read out from its genome sequence according to the SNARE hypothesis with a predictive accuracy of about 99.6%.
Proceedings of the National Academy of Sciences 05/2002; 99(8):5424-9. · 9.68 Impact Factor
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ABSTRACT: Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.
Journal of Cellular Biochemistry 02/2002; 85(1):101-11. · 2.87 Impact Factor
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ABSTRACT: Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in thetrans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution
of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated
using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6)
but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes
and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that
CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that
CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the
present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating
that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these
data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory
vesicles.
Journal of Biological Chemistry 05/1999; 274(21):14759-14767. · 4.77 Impact Factor
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ABSTRACT: Advisor: Lloyd D. Fricker. Thesis (Ph. D.)--Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, 1998. Includes bibliographical references (leaves 134-153). Typescript.
