The four-transmembrane domain proteins synaptophysin and synaptogyrin represent the major constituents of synaptic vesicles. Our previous studies in PC12 cells demonstrated that synaptogyrin or its nonneuronal paralog cellugyrin targets efficiently to synaptic-like microvesicles (SLMVs) and dramatically increases the synaptophysin content of SLMVs (Belfort, G. M., and Kandror, K. V. (2003) J. Biol. Chem. 278, 47971-47978). Here, we explored the mechanism of these phenomena and found that ectopic expression of cellugyrin increases the number of SLMVs in PC12 cells. Mutagenesis studies revealed that cellugyrin's hydrophilic cytoplasmic domains are not involved in vesicle biogenesis, whereas small conserved hydrophobic hairpins in the first luminal loop and the carboxyl terminus of cellugyrin were found to be critical for the formation of SLMVs. In addition, the length but not the primary sequence of the second luminal loop was essential for SLMV biogenesis. We suggest that changing the length of this loop similar to disruption of the short hydrophobic hairpins alters the position of the vicinal transmembrane domains that may be crucial for protein function.