Retention of Unassembled Components of Integral Membrane Proteins by Calnexin

Rajagopalan, Sumati; Xu, Yuhui; Brenner, Michael B.

doi:10.1126/science.8278814

Cited by 237 publications

(175 citation statements)

References 25 publications

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“…Orai1-YFP was clearly visible in the PM of spreading lamellipodia, and was also seen in moving puncta near the stimulatory surface ( Figure 1A, Movie 1). In Jurkat T-cells expressing STIM1-CFP plated onto coverslips coated with anti-CD45 antibodies that do not activate the TCR, STIM1-CFP was seen in an ER-like pattern that colocalized with ER markers such as calnexin (Rajagopalan et al, 1994) as expected from previous studies ( Figure 1B; Wu et al, 2006;Liou et al, 2007;Ross et al, 2007). Activation of these cells resulted in formation of puncta containing STIM1-CFP (Supplemental Figure S3).…”

Section: After Tcr Activation Orai1-yfp and Stim1-cfp Are Seen In Pusupporting

confidence: 56%

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

Barr

Bernot

Srikanth

et al. 2008

MBoC

129

142

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The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca 2؉ influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca 2؉ channel components to existing and newly forming immunological synapses. INTRODUCTIONT-cell activation in response to antigen induces and requires the elevation of intracellular Ca 2ϩ (Hogan et al., 2003;Quintana et al., 2005;Feske, 2007). T-cell receptor (TCR) engagement leads to activation of well-documented signal transduction pathways that cause a rapid release of Ca 2ϩ from the endoplasmic reticulum (ER; Samelson, 2002;Panyi et al., 2004;Gwack et al., 2007a). The depletion of Ca 2ϩ from this internal store then leads to the opening of ion channels in the plasma membrane (PM) allowing an influx of Ca 2ϩ from outside the cell. The store-operated channel responsible for Ca 2ϩ influx in T-cells is known as the Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel, which has been studied by electrophysiological techniques for many years (Lewis, 2001;Prakriya and Lewis, 2003;Cahalan et al., 2007;Hewavitharana et al., 2007;Hogan and Rao, 2007;Putney, 2007a).Sustained elevation of cytosolic Ca 2ϩ is required for complete T-cell activation (Iezzi et al., 1998;Lewis, 2001;Hogan et al., 2003;Feske, 2007). High levels of intracellular Ca 2ϩ are necessary to maintain the interaction between a T-cell and antigen-presenting cell (APC) that leads to formation of the specialized contact surface known as the immunological synapse (IS). Increased Ca 2ϩ levels are also required for the activation of transcription factors. In particular, elevated Ca 2ϩ maintains prolonged nuclear accumulation of nuclear factor of activated T-cells (NFAT) by activating the phosphatase calcineurin that dephosphorylates NFAT, allowing it to translocate to the nucleus and activate genes such as IL2 (Hogan et al., 2003;Macian, 2005). Several hours of Ca 2ϩ influx are required to complete the T-cell activation program, which involves expression of a large number of activation-associated genes (Lewis, 2001;Macian et al., 2002;Hogan et al., 2003).Although sustained Ca 2ϩ influx in T-cells has been studied for decades, the proteins involved have only been ident...

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Section: After Tcr Activation Orai1-yfp and Stim1-cfp Are Seen In Pusupporting

confidence: 56%

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

Barr

Bernot

Srikanth

et al. 2008

MBoC

129

142

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“…Indeed, immature CD4 Ϫ CD8 Ϫ thymocytes were recently found to express surface receptor complexes comprised of CD3␥ and CD3␦ heterodimers complexed with the molecular chaperone calnexin (24)(25)(26)(27)(28). This finding was remarkable because calnexin had never previously been found on the cell surface and because calnexin, CD3␥, and CD3␦ chains all have ER retention signals near their C termini (29,30). The calnexin-CD3 complexes that escape to the cell surface appear to do so because interactions between the cytoplasmic domains of calnexin and CD3 sterically mask their retention sequences, as has been reported for subunits of the immunoglobulin E receptor (24,31).…”

mentioning

confidence: 65%

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones

Wiest

Bhandoola

Punt

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The folding and assembly of nascent proteins in the endoplasmic reticulum (ER) is assisted by molecular chaperones that are themselves retained within the ER. We now report that a number of different ER proteins, including molecular chaperones, are selectively expressed on the surface of immature thymocytes, but their surface expression is extinguished upon further differentiation. Escape from the ER is only possible for newly synthesized ER proteins before they become permanently retained. Thus, the cellular process of ER retention is incomplete in immature thymocytes and provides an explanation for surface expression of partial receptor complexes that transduce differentiative signals during thymic development.

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“…For double immunofluorescence staining, the same procedure was repeated using monoclonal anti-calnexin primary antibodies provided by Affinity Bioreagents Inc. This antibody was raised against a human calnexin protein, a typical ER resident protein classically used as a marker for this organelle [20]. In this case, polyclonal anti-mouse IgG rhodamine-conjugated antibodies (Sigma) were used as secondary antibodies.…”

Section: Immunoftuorescence Microscopymentioning

confidence: 99%

Expression of a functionally active human hepatic UDP‐glucuronosyltransferase (UGT1A6) lacking the N‐terminal signal sequence in the endoplasmic reticulum

et al. 1999

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UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6Asp) was targeted to the endoplasmic reticulum in mammalian ceils as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wildtype. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGTIA6 suggesting that additional topogenic element(s) mediate(s) this process.

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Retention of Unassembled Components of Integral Membrane Proteins by Calnexin

Cited by 237 publications

References 25 publications

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones

Expression of a functionally active human hepatic UDP‐glucuronosyltransferase (UGT1A6) lacking the N‐terminal signal sequence in the endoplasmic reticulum

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