Proteomic Analysis of Two Functional States of the Golgi Complex in Mammary Epithelial Cells

Wu, Christine C.; Yates, John R.; Neville, Margaret C.; Howell, Kathryn E.

doi:10.1034/j.1600-0854.2000.011004.x

Cited by 75 publications

(38 citation statements)

References 58 publications

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“…Lactoferrin is also thought to contribute to iron acquisition in the neonate (16 -18, 21). Humans and monkeys have high levels of lactoferrin, and lactoferrin has been identified in rat mammary gland during the lactation period (36). High levels of lactoferrin receptors have been found in rhesus monkeys (5) and in human fetuses and infants (18).…”

Section: Discussionmentioning

confidence: 99%

Iron absorption by Belgrade rat pups during lactation

Thompson

Molina²,

Donaghey³

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

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valent metal transporter-1 (DMT1) mediates dietary nonheme iron absorption. Belgrade (b) rats have defective iron metabolism due to a mutation in the DMT1 gene. To examine the role of DMT1 in neonatal iron assimilation, b/b and b/ϩ pups were cross-fostered to F344 Fischer dams injected with 59 FeCl3 twice weekly during lactation. Tissue distribution of the radioisotope in the pups was determined at weaning (day 21). The b/b pups had blood 59 Fe levels significantly lower than b/ϩ controls but significantly higher 59 Fe tissue levels in heart, bone marrow, skeletal muscle, kidney, liver, spleen, stomach, and intestines. To study the pharmacokinetics of nonheme iron absorption at the time of weaning, 59 FeCl3 was administered to 21-day-old b/b and b/ϩ rats by intragastric gavage. Blood 59 Fe levels measured 5 min to 4 h postgavage were significantly lower in b/b rats, consistent with impaired DMT1 function in intestinal iron absorption. Tissue 59 Fe levels were also lower in b/b rats postgavage. Combined, these data suggest that DMT1 function is not essential for iron assimilation from milk during early development in the rat. iron transport; lactation; DMT1; Belgrade rat IRON IS ESSENTIAL FOR THE development and growth of the infant. The neonate receives its iron from milk in the form of nonheme iron (21) that has a high bioavailability (25,30). The molecular mechanisms responsible for the assimilation of iron from breast milk are not well understood. In the mature intestine, iron uptake across the apical membrane of the intestinal enterocyte is thought to be mediated by divalent metal transporter-1 (DMT1/SLC11A2). Mice with selective inactivation of DMT1 in the intestine (Slc11a2 int/int ) have normal hemoglobin and liver nonheme iron levels at birth, but the hematological parameters become reduced by 4 wk of age (15). Iron deficiency becomes even more pronounced after weaning (15). Although the metabolic defects observed in the Slc11a2 int/int mice support a significant role for DMT1 in intestinal iron uptake, whether targeted disruption of the DMT1 gene significantly impairs iron absorption during the early lactational feeding period is unclear. Immunohistochemical analyses have shown that minimal levels of DMT1 protein are present in the mouse intestine at postnatal days 0 to 5 (24). By postnatal day 10, DMT1 becomes localized in the apical membrane of the maturing intestine, but it is predominantly expressed in its deglycosylated/inactive form until postnatal day 20 (24). In the rat, levels of DMT1 expression continue to increase by postnatal day 40 (20), but the precise mechanisms responsible for the delivery of milk-bound iron in rats remain poorly understood. For humans, iron absorption from milk is thought to involve lactoferrin (16 -18, 21), but whether this milk protein is involved in iron absorption by rat pups is uncertain, particularly since rat enterocytes do not express lactoferrin receptors (17). An alternate pathway for iron uptake mediated by transferrin present in rat milk has been proposed (...

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Section: Discussionmentioning

confidence: 99%

Iron absorption by Belgrade rat pups during lactation

Thompson

Molina²,

Donaghey³

et al. 2007

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Samples were either first resolved by gel electrophoresis followed by gel excision, digestion, and extraction of both tryptic and non-tryptic (elastase digests) peptides (28,29) or directly digested in solution to produce both tryptic and non-tryptic peptides (30). Peptides prepared using both methods were analyzed by multidimensional protein identification technology (MudPIT) (31) with a Finnigan LTQ linear ion trap (ThermoElectron, San Jose, CA) using an in-house constructed microspray ion source.…”

Section: Methodsmentioning

confidence: 99%

Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C

Miranda

Sorkina

et al. 2005

Journal of Biological Chemistry

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120

133

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Dopamine transporter (DAT) localization in dopaminergic neurons plays an important role in regulating dopamine signaling. However, the mechanisms of DAT trafficking that control DAT localization are still poorly understood. To gain insight into these mechanisms, human DAT was purified in large amounts using a two-step affinity chromatography procedure from untreated HeLa cells or cells treated with phorbol 12-myristate 13-acetate (PMA). Mass spectrometric analysis of purified DAT complexes revealed the presence of several proteins, among which ubiquitin was particularly abundant in the PMA-treated sample. Western blotting of highly purified DAT protein confirmed constitutive ubiquitylation of DAT and a dramatic increase in DAT ubiquitylation in cells treated with PMA. This increase was blocked by pretreatment with the protein kinase C (PKC) inhibitor bis-indolylmaleimide. DAT ubiquitylation by ectopically expressed ubiquitin was demonstrated in cells transiently transfected with yellow fluorescent proteintagged ubiquitin. In addition, fluorescence resonance energy transfer was detected between cyan fluorescent protein-tagged DAT and yellow fluorescent protein-tagged ubiquitin, indicative of DATubiquitin conjugation. Interestingly, the largest fluorescence resonance energy transfer signals were observed in endosomes. Ubiquitylated DAT was detected in the plasma membrane using cell surface biotinylation as well as in intracellular compartments, suggesting that ubiquitylation begins at the plasma membrane and is maintained in endosomes. In both porcine aortic endothelial and HeLa cells, where PKC-dependent DAT ubiquitylation was observed, PKC activation resulted in rapid degradation of DAT (t1 ⁄ 2 ‫؍‬ 1-2 h). Altogether, these data suggest that PKC-induced DAT ubiquitylation may target DAT to lysosomal degradation. The dopamine transporter (DAT)2 is a member of the family of Na ϩ / Cl Ϫ -dependent plasma membrane transporters (SLC6 gene family) that are responsible for rapid clearance of neurotransmitters from the extracellular space. This family also includes norepinephrine, serotonin, ␥-aminobutyric acid, and glycine transporters (1). Members of the SLC6 family share similar predicted topology of a single polypeptide that contains twelve transmembrane segments, a large second extracellular glycosylated loop, and cytoplasmic amino-and carboxyl-terminal tails (2, 3). DAT and several other transporters of this family have been found to be constitutively oligomerized in vitro and in cells (4 -8).The amount of DAT at the plasma membrane and, therefore, dopamine uptake capacity are determined by trafficking of the DAT protein, which is regulated by several signaling cascades including signaling through protein kinase C (PKC). Activation of PKC by 4␣-phorbol 12-myristate 13-acetate (PMA) leads to a reduction in the V max of dopamine transport without a change in the substrate affinity (K m ) as well as in down-regulation of surface DAT protein (9 -15). This downregulation of DAT activity and levels has been shown to be ...

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“…The mammalian Golgi undergoes changes during differentiation or cancer progression (Kellokumpu et al, 2002;Kirk et al, 2010;Lu et al, 2001;Weller et al, 2010;Wu et al, 2000). In most eukaryotes, Golgi cisternae are arranged in polarized stacks, but in some species such as the yeast Saccharomyces cerevisiae, Golgi cisternae are dispersed throughout the cell (Mowbrey and Dacks, 2009;Preuss et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

Bhave

Papanikou

Iyer

et al. 2013

Journal of Cell Science

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Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1D cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1D mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number.

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Proteomic Analysis of Two Functional States of the Golgi Complex in Mammary Epithelial Cells

Cited by 75 publications

References 58 publications

Iron absorption by Belgrade rat pups during lactation

Iron absorption by Belgrade rat pups during lactation

Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C

Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation

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