The intracellular seven amino acid motif EEGEVFL is required for matriptase vesicle sorting and translocation to the basolateral plasma membrane

I− is actively transported into thyrocytes via the Na+/I− symporter (NIS), a key glycoprotein located on the basolateral plasma membrane. The cDNA encoding rat NIS was identified in our laboratory, where an extensive structure/function characterization of NIS is being conducted. Several NIS mutants have been identified as causes of congenital I− transport defect (ITD), including V59E NIS. ITD is characterized by low thyroid I− uptake, low saliva/plasma I− ratio, hypothyroidism, and goiter and may cause mental retardation if untreated. Studies of other ITD-causing NIS mutants have revealed valuable information regarding NIS structure/function. V59E NIS was reported to exhibit as much as 30% of the activity of wild-type NIS. However, this observation was at variance with the patients’ phenotype of total lack of activity. We have thoroughly characterized V59E NIS and studied several amino acid substitutions at position 59. We demonstrated that, in contrast to the previous report, V59E NIS is inactive, although it is properly targeted to the plasma membrane. Glu and all other charged amino acids or Pro at position 59 also yielded nonfunctional NIS proteins. However, I− uptake was rescued to different degrees by the other substitutions. Although the Km values for Na+ and I− were not altered in these active mutants, we found that the structural requirement for NIS function at position 59 is a neutral, helix-promoting amino acid. This result suggests that the region that contains V59 may be involved in intramembrane helix-helix interactions during the transport cycle without being in direct contact with the substrates.

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Regulation of Amino Acid Influx and Efflux at the Basolateral Plasma Membrane of the Salivary Gland Epithelium: Effects of Parasympathetic Nerve Stimulation

Journal of Dental Research ◽

10.1177/00220345870660023101 ◽

1987 ◽

Vol 66 (2) ◽

pp. 569-575 ◽

Cited By ~ 6

Author(s):

G.E. Mann ◽

D.L. Yudilevich

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Amino Acid ◽

Nerve Stimulation ◽

Transport Systems ◽

Acid Uptake ◽

Parasympathetic Nerve ◽

Large Neutral Amino Acids ◽

Neutral Amino Acids ◽

Basolateral Plasma Membrane

Basolateral amino acid transport systems in the salivary epithelium of resting and secreting cat submandibular glands were characterized by means of a rapid paired-tracer dilution technique. Amino acid uptake was measured by comparison of venous tracer concentration profiles for a labeled amino acid and D-mannitol (an extracellular tracer of similar size) following an intra-arterial bolus injection of both radioactive molecules. Unidirectional uptake of 21 amino acids, dopamine, noradrenaline, and serotonin was quantified in non-secreting glands. During 8 Hz parasympathetic nerve stimulation, significant epithelial uptakes were measured for L-[3H] alanine and L-[3H] phenylalanine, but less than 0.2% of the injected amino acid was recovered in the collected saliva. In non-secreting glands, cross-inhibition studies of L-[3H] alanine, L-[3H] phenylalanine, and L-[3H] lysine uptake by unlabeled amino acid competitors and detailed kinetic influx experiments indicated that epithelial uptake was mediated by three distinct parallel transport systems: ASC (short-chain neutral), L (branched-chain and aromatic neutral), and y+ (cationic). Rapid metabolism of alanine was inhibited by aminooxyacetate, and the metabolic uncoupler dinitrophenol selectively accelerated the efflux of transported large neutral amino acids and L-lysine. Concurrent autoradiographic experiments suggest that transport sites for small and large neutral amino acids are localized in the basolateral plasma membrane of acinar, demilunar, and striated ductal cells.

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Eps15 Mediates Vesicle Trafficking from thetrans-Golgi Network via an Interaction with the Clathrin Adaptor AP-1

Molecular Biology of the Cell ◽

10.1091/mbc.e07-10-0997 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3564-3575 ◽

Cited By ~ 28

Author(s):

Susan Chi ◽

Hong Cao ◽

Jing Chen ◽

Mark A. McNiven

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Adaptor Proteins ◽

Secretory Process ◽

Coated Vesicle ◽

Secretory Proteins ◽

Vesicle Formation ◽

Amino Acid Motif ◽

Clathrin Adaptor ◽

Golgi Network

Eps15 (EGFR pathway substrate clone 15) is well known for its role in clathrin-coated vesicle formation at the plasma membrane through interactions with other clathrin adaptor proteins such as AP-2. Interestingly, we observed that in addition to its plasma membrane localization, Eps15 is also present at the trans-Golgi network (TGN). Therefore, we predicted that Eps15 might associate with clathrin adaptor proteins at the TGN and thereby mediate the formation of Golgi-derived vesicles. Indeed, we have found that Eps15 and the TGN clathrin adaptor AP-1 coimmunoprecipitate from rat liver Golgi fractions. Furthermore, we have identified a 14-amino acid motif near the AP-2–binding domain of Eps15 that is required for binding to AP-1, but not AP-2. Disruption of the Eps15–AP-1 interaction via siRNA knockdown of AP-1 or expression of mutant Eps15 protein, which lacks a 14-amino acid motif representing the AP-1 binding site of Eps15, significantly reduced the exit of secretory proteins from the TGN. Together, these findings indicate that Eps15 plays an important role in clathrin-coated vesicle formation not only at the plasma membrane but also at the TGN during the secretory process.

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