Determinants in the ß and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-ß and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-ß and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, ßK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined ßK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the ß and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

Golgi localization of Syne-1.

We have previously identified a Golgi-localized spectrin isoform by using an antibody to the beta-subunit of erythrocyte spectrin. In this study, we show that a screen of a lambdagt11 expression library resulted in the isolation of an approximately 5-kb partial cDNA from a Madin-Darby bovine kidney (MDBK) cell line, which encoded a polypeptide of 1697 amino acids with low, but detectable, sequence homology to spectrin (37%). A blast search revealed that this clone overlaps with the 5' end of a recently identified spectrin family member Syne-1B/Nesprin-1beta, an alternately transcribed gene with muscle-specific forms that bind acetylcholine receptor and associate with the nuclear envelope. By comparing the sequence of the MDBK clone with sequence data from the human genome database, we have determined that this cDNA represents a central portion of a very large gene ( approximately 500 kb), encoding an approximately 25-kb transcript that we refer to as Syne-1. Syne-1 encodes a large polypeptide (8406 amino acids) with multiple spectrin repeats and a region at its amino terminus with high homology to the actin binding domains of conventional spectrins. Golgi localization for this spectrin-like protein was demonstrated by expression of epitope-tagged fragments in MDBK and COS cells, identifying two distinct Golgi binding sites, and by immunofluorescence microscopy by using several different antibody preparations. One of the Golgi binding domains on Syne-1 acts as a dominant negative inhibitor that alters the structure of the Golgi complex, which collapses into a condensed structure near the centrosome in transfected epithelial cells. We conclude that the Syne-1 gene is expressed in a variety of forms that are multifunctional and are capable of functioning at both the Golgi and the nuclear envelope, perhaps linking the two organelles during muscle differentiation.

Spectrins and the Golgi.

Several isoforms of spectrin membrane skeleton proteins have been localized to the Golgi complex. Golgi-specific membrane skeleton proteins associate with the Golgi as a detergent-resistant cytoskeletal structure that likely undergoes a dynamic assembly process that accommodates Golgi membrane dynamics. This review discusses the potential roles for this molecule in Golgi functions. In particular, it will focus on a recently identified distant cousin to conventional erythroid spectrin variously named Syne-1, Nesprin, myne, Enaptin, MSP-300, and Ank-1. Syne-1 has the novel ability to bind to both the Golgi and the nuclear envelope, a property that raises several intriguing and novel insights into Golgi structure and function. These include (1) the facilitation of interactions between Golgi and transitional ER sites on the nuclear envelope of muscle cells, and (2) an ability to impart localized specificity to the secretory pathway within large multinucleate syncytia such as skeletal muscle fibers.

The spectrin family member Syne-1 functions in retrograde transport from Golgi to ER.

To address the function of the Golgi- and nuclear envelope-localized spectrin family member synaptic nuclear envelope protein-1 (Syne-1), we expressed two separate recombinant fragments derived from the central portion of the molecule. Both of these fragments were predicted to act as dominant negative inhibitors of Syne-1 function at the Golgi. One of the fragments was previously shown to bind the Golgi complex. The other fragment was found to form microtubule-associated puncta that sequester endogenous Syne-1. Expression of either fragment resulted in a cell type-specific alteration in the structure of the Golgi complex, which appeared to collapse into a compact juxtanuclear structure in some cell types but not others. These fragments were expressed in cultured cells and their effects on Golgi function were examined. Expression of both dominant negative Syne-1 fragments blocked recycling of the endoplasmic reticulum (ER) resident protein disulfide isomerase (PDI), which accumulated in the Golgi complex. In addition, we found that fragment expression altered the distribution of the KDEL receptor and the COP-I coat protein beta-COP, two proteins known to be involved in regulating the retrograde pathway. We conclude that these results indicate a role for Syne-1 in facilitating retrograde vesicular trafficking from the Golgi to the ER.

A role for the spectrin superfamily member Syne-1 and kinesin II in cytokinesis.

Expression of a dominant negative fragment of the spectrin family member Syne-1 causes an accumulation of binucleate cells, suggesting a role for this protein in cytokinesis. An association of this fragment with the C-terminal tail domain of the kinesin II subunit KIF3B was identified by yeast two-hybrid and co-precipitation assays, suggesting that the role of Syne-1 in cytokinesis involves an interaction with kinesin II. In support of this we found that (1) expression of KIF3B tail domain also gives rise to multinucleate cells, (2) both Syne-1 and KIF3B localize to the central spindle and midbody during cytokinesis in a detergent resistant and ATP sensitive manner and (3) Syne-1 localization is blocked by expression of KIF3B tail. Also, membrane vesicles containing syntaxin associate with the spindle midbody with identical properties. We conclude that Syne-1 and KIF3B function together in cytokinesis by facilitating the accumulation of membrane vesicles at the spindle midbody.