Monoubiquitination of Stx3 leads to efficient endocytosis from the basolateral plasma membrane and trafficking into the multivesicular body/exosomal pathway. Stx3 plays a role in cargo recruitment into exosomes. This pathway is exploited by HCMV for virion excretion.

Plasma cells (PC) are B-lymphocytes terminally differentiated in a postmitotic state, with the unique purpose of manufacturing and exporting Igs. Despite the importance of this process in the survival of vertebrates, no studies have been made to understand the molecular events that regulate Ig exocytosis by PC. The present study explores the possible presence of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) system in human PC, and examines its functional role in Ig secretion. Syntaxin-2, Syntaxin-3, Syntaxin-4, vesicle-associated membrane protein (VAMP)-2, VAMP-3, and synaptosome-associated protein (SNAP)-23 could be readily detected in normal human PC obtained from intestinal lamina propria and blood, as well as in human PC lines. Because SNAP-23 plays a central role in SNAREs complex formation, it was chosen to examine possible functional implications of the SNARE system in PC Ig secretion. When recombinant SNAP-23 fusion protein was introduced into the cells, a complete abolishment of Ig production was observed in the culture supernatants of PC lines, as well as in those of normal PC. These results provide insights, for the first time, into the molecular machinery of constitutive vesicular trafficking in human PC Ig secretion and present evidence indicating that at least SNAP-23 is essential for Ab production.

B-lymphocyte-induced maturation protein-1 (BLIMP1), encoded by the PRDM1 gene, is a transcriptional repressor considered a master regulator that is required and sufficient for plasma cell (PC) differentiation. BLIMP1 represses the PAX5 gene, coding for the B-cell lineage-specific activator protein (BSAP), which is required for B-cell identity and survival. Mutations in PAX5 gene as well as in PRDM1 gene have been recently implicated in lymphomas. In the present study, sequence analysis of PRDM1 gene revealed a binding site for BSAP transcription factor. By analyzing different human cell lines, we have found that a specific nuclear factor for B-cell lines binds to a site on the PRDM1 promoter. Electrophoretic mobility shift assays identified this factor as BSAP, and chromatin immunoprecipitation assays confirmed its binding in vivo to the human PRDM1 promoter. Moreover, by ectopically expressing BSAP, and using a IntroductionIn multicellular organisms, cell development and cell differentiation into many cell types is under the control of transcriptional factors. In the vertebrate immune system, the B-lymphocyte developmental pathway represents a model for the analysis of genetic networks, which orchestrate cell fate specification and commitment. Plasma cell (PC) differentiation from B lymphocyte depends on the switch-on/-off balance between transcription factors. 1,2 In this context, BSAP is a critical transcription factor required to establish and maintain B-cell lineage identity until the PC stage. [3][4][5] BSAP is a bifunctional transcription factor that can, depending on the gene context, either activate transcription of genes involved in maintaining B-cell identity including CD19, 6 Ig␣, BLNK, and CIITA 4 or repress transcription of PC-associated gene commitment such as J chain, IgH, IgL, and XBP1. [7][8][9][10] In the absence of PAX5, the gene coding for BSAP, progenitor B cells acquire the ability to differentiate into multiple hematopoietic lineages both in vitro and in vivo. [11][12][13] On the other hand, overexpression of PAX5 in the late stage of B-cell lines and in PC lines leads to increased cell proliferation and suppression of Ig synthesis. 14 Thus, repression of PAX5 is important for inhibiting B-lymphocyte functions and is required for PC functions. 15,16 Moreover, recent studies demonstrate the oncogenic role of BSAP by mutations or alterations in the expression of PAX5 gene. [17][18][19][20][21] Murine B-lymphocyte-induced maturation protein-1 (BLIMP1), or its human homologue positive regulatory domain I binding factor 1 (PRDI-BF1), is a transcription factor that has been demonstrated to act as a master regulator required 10 and sufficient 22 for the generation and for the prolonged maintenance of PCs. 23 To differentiate into PCs, BLIMP1 reduces B-cell proliferation as a consequence of MYC repression, 24 and decreases B-cell functions as a result of PAX5 repression. 25 An essential gene for PC differentiation indirectly induced by BLIMP1, as well as by IRF4, is XBP1. [25][26][27] Moreover, ...

The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension.

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24–29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.