Cohen syndrome-associated protein, COH1, is a novel, giant Golgi matrix protein required for Golgi integrity

Loss-of-function mutations in the gene COH1, also known as VPS13B, lead to autosomal recessive Cohen syndrome. However, the cellular distribution and function of the encoded protein COH1 (3997 amino acids), which lacks functional homologies to other mammalian proteins, have remained enigmatic. We show here that COH1 is a peripheral Golgi membrane protein that strongly co-localizes with the cis-Golgi matrix protein GM130. Consistent with its subcellular localization, COH1 depletion using RNAi causes fragmentation of the Golgi ribbon into ministacks. Disruption of Golgi organization observed in fibroblasts from Cohen syndrome patients suggests that Golgi dysfunction contributes to Cohen syndrome pathology. In conclusion, our findings establish COH1 as a Golgi-associated matrix protein required for Golgi integrity.     

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.     

Actinfilin,a brain-specific actin-binding protein in postsynaptic density

The dynamic assembly and disassembly of actin-based cytoskeleton is closely linked to the changes in the postsynaptic density in both number and shape, which is thought to be important in forming long-term memory. Thus, regulation of actin filaments may play a critical role in contributing to the formation of long-term memory. Here, we report the cloning of actinfilin, a brain-specific Kelch protein, which interacts with F-actin. Actinfilin contains an amino-terminal POZ/BTB domain and carboxyl positioned six tandem Kelch repeats that presumably form six blades of beta-propeller structure of the Kelch domain. Co-immunoprecipitation analyses showed that the amino-terminal POZ domain mediated actinfilin-actinfilin interaction. The recombinant Kelch domain alone was sufficient to mediate binding to F-actin. Immunohistochemistry studies of rat brain sections suggested that actinfilin is broadly expressed in neurons of most regions of the brain. The subcellular localization of actinfilin was studied by biochemical fractionation and immunogold labeling. The results showed the postsynaptic density distribution of actinfilin. Together, these results indicate that actinfilin may be a key player in the actin-based neuronal function.     

Characterization of a cis-Golgi matrix protein, GM130

Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER- Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.     

Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH(2)-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide-sensitive fusion protein)-catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of trans-SNARE [corrected] (trans-soluble NSF attachment protein [SNAP] receptor) complexes.     

The Membrane Transport Factor TAP/p115 Cycles between the Golgi and Earlier Secretory Compartments and Contains Distinct Domains Required for Its Localization and Function

The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al., 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15°C treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi.TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH₂-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH₂-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.     

BTB/POZ domain of speckle-type POZ protein (SPOP) confers proapoptotic function in HeLa cells

The proapoptotic function of SPOP protein was investigated in HeLa cells. HeLa cells underwent apoptosis by the overexpression of SPOP. Studies using SPOP deletion mutants suggest that BTB/POZ domain of SPOP protein is important for the induction of apoptosis in transfected cells. This study first proposes the proapoptotic aspect of the BTB/POZ domain of SPOP protein based on the finding that cells expressing the C-terminal fragment of SPOP containing the BTB/POZ domain underwent apoptosis.     

Identification of Nd1, a novel murine kelch family protein,involved in stabilization of actin filaments

We isolated Nd1, a novel kelch family gene that encodes two forms of proteins, Nd1-L and Nd1-S. Nd1-L contains a BTB/POZ domain in its N terminus and six kelch repeats in the C terminus. Nd1-S has the BTB/POZ domain but lacks the six kelch repeats. Nd1-L but not Nd1-S mRNA is detected ubiquitously in normal mouse tissues. Nd1-L and Nd1-S proteins can form a dimer through the BTB/POZ domain. Nd1-L colocalizes with actin filaments detected using a confocal microscope, and its kelch repeats bind to them in vitro. Overexpression of Nd1-L in NIH3T3 cells delayed cell growth by affecting the transition of cytokinesis. Furthermore, the overexpression prevented NIH3T3 cells from cell death induced by actin destabilization but not by microtubule dysfunction. These data suggest that Nd1-L functions as a stabilizer of actin filaments as an actin-binding protein and may play a role in the dynamic organization of the actin cytoskeleton.